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Full text of "The biology of pneumococcus; the bacteriological, biochemical, and immunological characters and activities of Diplococcus pneumoniae"

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THE BIOLOGY OF 
PNEUMOCOCCUS 



THE BIOLOGY OF PNEUMOCOCCUS 

THE BACTERIOLOGICAL, BIOCHEMICAL, AND 

IMMUNOLOGICAL CHARACTERS AND ACTIVITIES 

OF DIPLOCOCCUS PNEUMONIAE 



BENJAMIN WHITE, PH.D. 

with the collaboration of 

ELLIOTT STIRLING ROBINSON, M.D., PH.D. 

AND LAVERNE ALMON BARNES, PH.D. 



1938 
NEW YORK ■ THE COMMONWEALTH FUND 
London ■ Humphrey Milford ■ Oxford University Press 



COPYRIGHT, 1938, BY 

THE COMMONWEALTH FUND 

PUBLISHED FEBRUARY 1938 

SECOND PRINTING, MARCH 1941 



THE COMMONWEALTH FUND, 41 EAST 57TH STREET, NEW YORK, N.Y. 

LITHOGRAPHED IN THE UNITED STATES OF AMERICA BY DUENEWALD 
PRINTING CORPORATION 



Affectionately dedicated to the memory of 

GEORGE HOYT BIGELOW, M.D. 

who gave to the Massachusetts Pneumonia Study 

and Service an inspiring guidance, and who, by 

his gallantry, set us an enduring and ennobling 

example of scientific fellowship 



FOREWORD 

In the introduction to one of his Lectures and Lay Sermons, 
Huxley used an analogy aptly illustrating his subject, and 
singularly fitting the purposes of the present undertaking: "Mer- 
chants occasionally go through a wholesome, though troublesome 
and not always satisfactory, process which they term 'taking 
stock.' After all the excitement of speculation, the pleasure of 
gain, and the pain of loss, the trader makes up his mind to face 
facts and to learn the exact quantity and quality of his solid and 
reliable possessions. The man of science does well sometimes to 
imitate this procedure; and, forgetting for the time the importance 
of his own small winnings, to re-examine the common stock in 
trade, so that he may make sure how far the store of the bullion in 
the cellar — on the faith of whose existence so much paper has been 
circulating — is really the solid gold of truth." 

The present undertaking is not an inventory of small personal 
winnings, but rather a re-examination of the circulating paper and 
an attempted appraisal of the quantity and quality of the stored 
bullion, done with a sense of humility before the importance and 
magnitude of the task. Nor is this an original enterprise. There 
are the familiar Rockefeller Institute Monograph, Number Seven, 
the ample summary of Neufeld and Schnitzer, and the later sym- 
posium in the British System of Bacteriology, but since their pub- 
lication more gold has been mined, vastly enriching the common 
stock. In drawing upon this stock for the purpose of learning bet- 
ter ways of applying our knowledge to the discovery of means for 
mitigating the ills inflicted upon man by Pneumococcus, the whole 
storehouse has been ransacked for hidden or forgotten goods. To 
sort out the accumulation of more than fifty years, to convert old 
specie into modern currency, and finally to set a value on the 
whole store has been a troublesome task which it is hoped may be 



viii BIOLOGY OF PNEUMOCOCCUS 

spared others by the presentation of this re-examination and ap- 
praisal. 

The present review of the literature on the biology of Pneumo- 
coccus forms a part of the Pneumonia Study and Service carried 
on during the years 1931 to 1935 by the Massachusetts Depart- 
ment of Public Health under a grant from the Commonwealth 
Fund. 

The labor of preparing the manuscript has been lightened and 
made an agreeable occupation by the help so graciously given by 
many friends. An added pleasure, now that the work has come to 
completion, is to record grateful acknowledgments to the officers 
and members of the staff of the Commonwealth Fund for their 
sympathetic interest and liberal support; to Professor Doctor Fr. 
Neufeld for permission to reproduce original drawings from his 
publications and for aid in securing some of the portraits included 
in the text ; to Dr. Rufus Cole and colleagues at the Hospital of 
the Rockefeller Institute for Medical Research for many cour- 
tesies ; to Dr. O. T. Avery for the rich benefits of his counsel ; to 
Dr. M. A. Dawson for the generous loan of unpublished illustra- 
tive material ; to Miss L. M. D. Trask, librarian of the Rocke- 
feller Institute, for her able assistance in searching the literature; 
to Miss E. C. Campbell of the Publication Division of the same 
Institute for the loan of original electrotypes from the Journal of 
Experimental Medicine; to Miss Emily Jackson and associates at 
the Massachusetts Antitoxin and Vaccine Laboratory for their 
competent and willing services ; to many of the authors quoted 
for criticism and suggestions ; and to Mrs. Helen M. Boynton for 
her intelligent and discriminating digest of the whole story of 
Pneumococcus. 

B. W. 
Boston, 1937 



CONTENTS 

I. HISTORY OF PNEUMOCOCCUS 1 

1875-1890: inoculation of rabbits with human sputum 
(Pasteur and Sternberg) ; isolation, cultivation, and ani- 
mal inoculation (Friedlander) ; pneumococci in dust and 
air (Emmerich and Pawlowsky) ; Fraenkel's experiments; 
acquired resistance to infection ; Weichselbaum's contri- 
butions ; artificial active immunity. 1890-1900: passive 
immunity (Klemperers) ; dissociation (Kruse and Pan- 
sini) ; agglutination (Metchnikoff and others) ; type dif- 
ferentiation ; tropins (Denys). Summary. 

II. BIOLOGY OF PNEUMOCOCCUS 30 

Morphology : staining methods ; the capsule ; staining the 
capsule. Isolation of Pneumococcus: animal inoculation; 
direct cultures. Cultivation: accessory substances; vege- 
table accessory substances; appearance of growth; differ- 
ential media. Viability. Autolysis. Bile solubility. Sensi- 
tiveness to germicides and other chemical substances. 

III. BIOCHEMICAL FEATURES 65 

Proteolysis, lipolysis, and carbohydrate fermentation. 
Acid production. Oxidation and reduction: methemoglobin 
production ; peroxide formation. Hemolysin and hemo- 
toxin. Purpura production. Virulin, leucocidin, and analo- 
gous substances. So-called toxins. Summary. 

IV. CLASSIFICATION OF PNEUMOCOCCI 103 

Serological classification, 1898-1932: first differentiation 
by agglutination reaction ; differentiation by immunologi- 
cal reactions; Pneumococcus mucosus; two groups; typi- 
cal and atypical strains ; four groups ; five groups ; sub- 
groups of Type II; Group IV differentiated into twelve 
groups; further differentiation of subgroups of Type II; 
further differentiation of subgroups of Group IV. Classi- 
fication according to electrophoretic potential. Type de- 
termination : mouse protection test ; culture agglutination ; 
urine precipitation test; sputum precipitinogen; sputum 
digestion; bile-solution of sputum; slide agglutination; 
Quellung phenomenon. Summary. 



x BIOLOGY OF PNEUMOCOCCUS 

V. PNEUMOCOCCAL DISSOCIATION AND TRANSFORMATION 134 

Early observations of dissociation, 1891-1921. Later ob- 
servations of dissociation: smooth and rough forms of 
Pneumococcus ; modifications A, B, and C ; composite cul- 
tures ; species-specificity of rough forms ; electrophoretic 
potential of variants ; effect of charcoal, yeast, optochin ; 
in vivo variation ; details of colony formation ; antigenicity 
of rough forms ; respiratory capacity of variants ; in- 
termediate forms ; reversal of dissociation ; reversion 
by means of pneumococcal vaccine. Transformation of 
type: transformation by vaccine and animal inoculation; 
isolation of the transformative principle. Dawson classifi- 
cation. Transmutation of species. Summary. 

VI. PATHOGENICITY OF PNEUMOCOCCUS: EXPERIMENTAL 
ANIMALS 179 

Susceptibility of the animal host: the rabbit; the guinea 
pig; the mouse; the rat; the monkey; the cat; the dog; 
the horse; birds. Virulence of the organism: freshly iso- 
lated strains; numbers of cocci required to infect; avenue 
of inoculation ; determination of virulence ; substances 
that enhance virulence ; cultural conditions and virulence ; 
strain variations in virulence; virulence in respect to ani- 
mal species; artificial exaltation of virulence; degradation 
of virulence; dissociation and virulence. Summary. 

VII. PATHOGENICITY OF PNEUMOCOCCUS: MAN 214 

Etiology of pneumonia : pneumococcal types in lobar 
pneumonia ; pneumococcal types in bronchopneumonia ; 
pneumococcal types in pneumonia in infants and children. 
Serological types and fatality-rates. Localized epidemics 
of pneumococcal infection. Infectious processes other 
than pneumonia. Pneumococcemia. Excretion of pneumo- 
cocci. The carrier state. Summary. 

VIII. CHEMICAL CONSTITUENTS OF PNEUMOCOCCUS 238 

Work of earlier investigators. First description of the 
pneumococcal carbohydrate. Isolation of carbohydrate 
fractions. Isolation of C Fraction. Relation of the pneu- 
mococcal carbohydrate to carbohydrates isolated from 
other organisms. Function of sugars in determining anti- 
genic specificity and conjugated proteins. Isolation of "A 
substance." Comparison of various cellular carbohydrates. 
Type VIII carbohydrate. Isolation of an unidentified con- 



CONTENTS xi 

stituent of the pneumococcal cell. Cause of differences in 
carbohydrates isolated from Pneumococcus. Relation of 
the acetyl group to the immunological activity of Pneumo- 
coccus. Recent methods of preparing capsular polysaccha- 
ride. Other physicochemical properties of the capsular 
polysaccharide. Summary. 

IX. SPECIFIC POLYSACCHARIDE-SPLITTING ENZYMES 301 

First isolation of polysaccharide-splitting enzyme: SI II 
bacillus. Effect of the enzyme on the cell capsule. Pro- 
tective action of enzyme in mice. Philosophical aspect of 
the action of the enzyme. Methods of production. Effect 
of enzyme on infection induced in rabbits. Effect on in- 
fection induced in monkeys. Isolation and study of other 
bacteria possessing polysaccharide-splitting enzymes. 
Differences in susceptibility of polysaccharides to en- 
zymatic action. Summary. 

X. ANTIGENICITY OF PNEUMOCOCCUS 323 

Antigenic spectrum. First observations of immunity. In- 
fluence of virulence on immunological response. Dead 
cultures: heat-killed antigens; other devitalized antigens. 
Sensitized pneumococci. Filtrates and extracts : culture 
filtrates and bacterial extracts ; tissue extracts and exu- 
dates. Toxins and hemotoxins: so-called toxins; hemo- 
toxins. Methods of administering antigens: intradermal 
injection; inhalation and intrabronchial insufflation; oral 
administration. Host response. Antagonistic action of sol- 
uble specific substance. Summary. 

XI. ANTIBODIES TO PNEUMOCOCCUS 355 

Agglutinins: history; agglutinins in the blood of pneu- 
monia patients ; agglutination and serological classifica- 
tion ; agglutinability of pneumococcal variants ; agglu- 
tinins in the blood of animals ; additional data. Precipi- 
tins : history ; antiprotein precipitins ; somatic carbohy- 
drate (C Fraction) ; the mechanism of specific precipita- 
tion; correlation of precipitins with other antibodies. 
Complement-fixing antibodies. Bactericidins. Antihemo- 
toxin. Antitoxin. Heterophile antibodies. Phagocytosis: 
history; sensitization of pneumococci; normal tropins or 
opsonins ; antiopsonins ; opsonins in pneumonia ; the 
mechanism of phagocytosis. Protective antibodies: speci- 
ficity ; relation to other antibodies ; additional data ; 



xii BIOLOGY OF PNEUMOCOCCUS 

chemical nature of protective antibodies; the separation 
of antibodies from immune serum; the estimation of pro- 
tective antibodies. Other immunological phenomena: 
growth of pneumococci in specific immune serum; anti- 
blastic immunity; immunological relationships between 
Pneumococcus and other microbic species and unrelated 
substances. Summary. 

XII. HOST RESPONSE TO ANTIGENIC ACTION OF PNEUMO- 
COCCUS 427 

Natural immunity: in animals; in man. Naturally induced 
immunity: specific antibodies in the blood during pneu- 
monia; immune substances in sputum; antagonistic sub- 
stances in pneumococcal exudates. Artificially induced 
immunity: active immunity; passive immunity. Allergy 
and anaphylaxis: actively induced sensitization; passive 
sensitization ; relation of pneumococcal allergy to pneu- 
mococcal immunity. Dermal allergy: skin reactions in ex- 
perimental animals; skin reactions in lobar pneumonia; 
active and passive skin allergy ; the mechanism of dermal 
allergy; the Shwartzman phenomenon. Summary. 

XIII. PNEUMOCOCCAL VACCINES 479 

Experiments on monkeys. Types of vaccines employed. 
Dosage. Special constituents of Pneumococcus. Potency 
tests on vaccines. Route and spacing of injections. Local 
and systemic reactions. Appearance and duration of vac- 
cinal immunity. Results following vaccination in man. 
Prophylaxis. Vaccine treatment of pneumonia. 

XIV. CHEMOTHERAPY 507 

Chemical agents other than cinchona derivatives: bile; 
soap; coal-tar dyes; metals and metallic salts; other me- 
dicinal agents. Cinchona derivatives : effect of the various 
quinine derivatives on pneumococci; effect on various 
pneumococcal types; effect on pneumococcal infection; 
adjuvant action of cinchona compounds with specific se- 
rum; effect on virulence of Pneumococcus. Summary. 

XV. PRODUCTION OF ANTIPNEUMOCOCCIC SERUM 522 

Immunization of the horse: selection of horses; selection 
and standardization of the immunizing antigen; injec- 
tions; bleedings. The production of therapeutic serum: 
reasons for and against the use of unconcentrated serum ; 



CONTENTS xiii 

avian serum; polyvalent serum; methods of concentrating 
serum. Physical properties. Chill-producing factors. Po- 
tency tests: in vivo tests; in vitro tests; correlation be- 
tween in vitro and in vivo tests. Safety tests: routine 
safety test; tests on mice. Final processing of serums: 
total solids ; filtration ; bulk sterility and potency tests ; 
dispensing and labeling; sterility tests on final contain- 
ers ; safety and identity tests ; identity test ; records ; 
regulations governing antipneumococcic serum. The pro- 
duction of diagnostic serum: immune horse serum; im- 
mune rabbit serum ; preservatives, bottling, and labeling. 
Summary. 

XVI. SERUM TREATMENT OF LOBAR PNEUMONIA 598 

The rationale of serum therapy. Problems and limitations 
of serum therapy. The results of serum therapy. Sum- 
mary. 

XVII. UNSOLVED PROBLEMS 613 

Toxins. Serological types. Dissociation. Chemical con- 
stituents of pneumococci. Virulence. Methods for the pro- 
duction of active immunity. Chemotherapy. Immunologi- 
cal response. Antibodies and animal species. Skin reac- 
tions. The nature of pneumococcal antibody. Concentra- 
tion of antipneumococcic serum. Potency tests on thera- 
peutic serum. Summary. 

APPENDIX. SPECIAL METHODS USED IN THE STUDY OF PNEU- 
MOCOCCUS AND IN THE PREPARATION OF ANTIPNEUMO- 
COCCIC SERUM 623 

i. media. Pneumococcus broth for all general purposes. 
Nutrient broth for mass cultures for the production of 
soluble specific substances: preparation of infusion; 
preparation of medium; growing the pneumococci. Blood 
broth. Inulin serum water. 623 

ii. isolation of pneumococcus. Mouse method: mouse in- 
oculation; mouse necropsy. Plating methods: preparation 
of blood agar; use of blood agar. Blood cultures. 626 

in. type determination. Mouse method. Krumwiede 
method. Neufeld Quellung method. Urine test: unconcen- 
trated urine ; concentrated urine for precipitin test. 628 

IV. ISOLATION OF SEVERAL COMPONENTS OF PNEUMOCOCCUS. 

Medium for the production of culture of standard maxi- 



BIOLOGY OF PNEUMOCOCCUS 

mal density, virulence, and polysaccharide content. Pneu- 
mococcal protein. Somatic carbohydrate or C Fraction. 
Capsular polysaccharide: preparation of the specific poly- 
saccharide of Type I Pneumococcus ; preparation of the 
specific polysaccharide of Type II Pneumococcus; prepa- 
ration of the specific polysaccharide of Type III Pneu- 
mococcus. 634 

V. PREPARATION OF BACTERIAL ENZYMES CAPABLE OF DECOM- 
POSING CAPSULAR POLYSACCHARIDES. 640 

vi. serological reactions. Agglutination. Precipitation: 
optimal proportions ; nitrogen determination ; modified 
routine; combining equivalents. Complement fixation. 
Bactericidal tests. 641 

VII. POTENCY TESTS ON ANTIPNEUMOCOCCIC SERUM. United 

States Hygienic Laboratory, now National Institute of 
Health : culture ; serum ; the test." League of Nations : ani- 
mals; culture; serum. American Drug Manufacturers' As- 
sociation: unit of serum; test dose of culture; the test; 
interpretation of test; records and reports. Massachu- 
setts Antitoxin and Vaccine Laboratory : culture ; serums ; 
the test; interpretation of the test; protocols and records. 648 

VIII. STERILITY TESTS ON ANTIPNEUMOCOCCIC SERUM. Bulk 

sterility tests. Sterility tests on final containers. 662 

IX. PREPARATION OF DIAGNOSTIC ANTIPNEUMOCOCCIC RABBIT 

serum. Massachusetts Antitoxin and Vaccine Laboratory 
method: vaccine; injection of rabbits; bleeding of rab- 
bits ; testing of serum ; dilution of serum. 663 



BIBLIOGRAPHY 



665 



INDEX 771 



INTRODUCTION 

Pneumococcus is altogether an amazing cell. Tiny in size, sim- 
ple in structure, frail in make-up, it possesses physiological 
functions of great variety, performs biochemical feats of extraor- 
dinary intricacy and, attacking man, sets up a stormy disease so 
often fatal that it must be reckoned as one of the foremost causes 
of human death. Furthermore, living or dead, whole or in part, on 
entering the animal body Pneumococcus starts a train of impulses, 
stimulating all the reactions grouped under those inclusive phe- 
nomena known as immunity. 

Digesting foreign proteins, Pneumococcus rebuilds the frag- 
ments into a new protein common to all types of the species ; split- 
ting and consuming carbohydrates, from the simplest sugars to 
the starch-like substances, inulin and glycogen, the cell synthesizes 
the cleavage products of these same sugars into complex poly- 
saccharides. These polysaccharides, chemically distinct and im- 
munologically specific for the type, are built into a morphological 
structure which forms a defensive armor against the destructive 
forces of the animal body, and in a highly selective fashion de- 
termines the precise nature of the immunological response each 
separate type calls forth. 

Man has not been content to allow Pneumococcus to destroy 
human life in an unrestrained way. The sanitarian has sought 
hygienic and prophylactic measures to curb the incidence of acute 
respiratory infections ; the pathologist has studied the anatomical 
distribution and histological nature of pulmonary lesions in order 
to gain fuller knowledge of the portal of entry and the pathway 
of infection through the living tissues ; the epidemiologist has at- 
tempted to track the mode of dissemination of the infectious agent 
in the hope of breaking the vicious cycle of transmission from man 
to man ; pending the discovery of specific therapy, the physician 
has ameliorated suffering by symptomatic treatment and con- 



xvi BIOLOGY OF PNEUMOCOCCUS 

tributed to recovery by instituting supportive measures until the 
natural capabilities of living tissue have repaired the injuries in- 
flicted by the invading microorganisms. 

The bacteriologist has persisted in the pursuit of information, 
and as a result the positive identification, the growth needs, and 
much of the physiological behavior of Pneumococcus are now com- 
mon knowledge. The immunologist, by serum reactions, has found 
that pneumococci are not all alike; that they may become de- 
graded and even transmuted into strange forms, and that on this 
degradation or exaltation depend certain vital processes within 
the cell related to virulence and the morbid effects produced in the 
animal economy. With this information he has devised ways of 
identifying types among the species, and of using the cocci for 
his own purposes. 

In time the chemist joined forces with his colleagues in the field 
of medical science, and as a result of their combined efforts the 
answer has been found to some of the riddles which the bacteri- 
ologist alone could not solve. Breaking down Pneumococcus into 
its component parts, the chemist found the expected protein, and 
then discovered the new sugars which have so altered our ideas 
about pneumococci in general, and which are bringing a solution 
to the cryptic working of the immunological mechanism. 

The chemist, laboring with the bacteriologist and immunologist, 
has given us a new conception of the significance of chemical con- 
stitution and molecular arrangement in determining the power of 
proteins and of carbohydrates, either alone or combined, to call 
into activity those reactions which spell the fate of the infected 
host or the fate, not only of Pneumococcus, but of other bacteria 
and even of non-living alien substances that gain entrance to the 
animal body. New biological principles have been disclosed, ex- 
plaining many of the functions of the living bacterial cell amidst 
both natural and artificial surroundings and opening fresh and 
inviting paths of investigation. 

The study of the members of this small group of microorganisms 



INTRODUCTION xvii 

in a subordinate branch of biology is bringing light into some of 
the obscure realms of the related sciences. The peculiarities of 
Pneumococcus are yielding a generous return to the investors and 
speculators who have cast in their resources with its lot, resulting 
in the accumulation of a store of solid bullion for the scientist 
and for mankind. 



THE BIOLOGY OF 
PNEUMOCOCCUS 








Photograph by Giraudon, Paris 

LOUIS PASTEUR 1 82 2-1 895 
By Louis Edoimrd Fournier, Ecole Norrnale Supdrieure, Paris 



CHAPTER I 
HISTORY OF PNEUMOCOCCUS 

A chronological account of the early observations of the coccus in 
tissues, saliva, and other body fluids; first isolation and cultiva- 
tion, and description of effects on animals; separation into types; 
variation; and attempts to produce immune serum. 

The lanceolate, Gram-positive diplococcus, now known as the 
chief etiological agent in lobar pneumonia and commonly 
called Pneumococcus, has the species name Diplococcus pneu- 
moniae, genus Diplococcaceae, tribe Streptococcaceae of the fam- 
ily Coccaceae. 10 * During the years in which the organism has been 
the subject of investigation, it has received many appellations 
which are given below in chronological order. 

Monas pulmonale (Klebs) 718 1875 

Microbe septicemique du saliva (Pasteur) 1066 1881 

Pneumoniekokken (Matray) 869 " 70 1883 

Coccus lanceole de la pneumonie (Talamon) 1377 1883 

Pneumoniemikrokokken (Friedlander) 487 1883 

Pneumonie-Micrococcen (Friedlander) 490 1884 

Micrococcus Pasteuri (Sternberg) 1319 1885 

Pneumoniemikrococcus (Fraenkel) 469 1886 

Pneumococcus (Fraenkel) 469 1886 

Bacillus septicus sputigenus (Fliigge) 495 1886 

Diplokokkus lanceolatus pneumoniae (Fliigge) 495 1886 

Diplococcus pneumoniae (Weichselbaum) 1497 1886 

Bacillus salivarius septicus (Biondi) 117 1887 

Micrococcus pneumoniae crouposae (Sternberg) 1320 1887 

Streptococcus lanceolatus Pasteuri (Gamaleia) 498 1888 

Diplococcus lanceolatus (Foa, Bordoni-Uffreduzzi) 462 1888 

Virus pneumonico (Gabbi) 497 1889 

Bacterium pneumoniae (Migula) 901 1900 

Diplococcus pneumoniae Weichselbaum (Bergey) 104 1930 

In giving proper credit for the discovery of an organism it usu- 



2 BIOLOGY OF PNEUMOCOCCUS 

ally becomes necessary to decide which special events constitute a 
discovery. Is it the report of the first sight of the organism in the 
fluids or tissues of the infected animal ; is it the first formal asso- 
ciation of the virus with a disease ; is it the setting up of a specific 
infection in an alien animal species ; or must one await the complete 
fulfilment of all the postulates of Koch's law? 

It is not essential to our purpose to be too exacting in asking or 
answering these questions. It should suffice to describe the observa- 
tions of early pioneers and their successors in the order of their 
happening. 

1875-1890 

In 1875, Klebs 718 was probably the first to see — or at least the 
first to tell about seeing — Pneumococcus. Searching for proof of 
the infectious nature of pneumonia, he examined fluid from the 
lungs of men dying of the disease and found non-motile, sometimes 
linked monads in astonishing numbers. Growing these cocci on egg 
albumen, he thought they developed motility when grouped in 
chains, but their true identity escaped him. In 1880, Eberth 347 was 
undoubtedly looking at pneumococci when he examined the exudate 
from a gray-hepatized lung and the subarachnoid fluid of a pneu- 
monia patient with a secondary meningitis. His description of the 
organisms as non-motile, slightly oval, almost round bodies, occa- 
sionally occurring singly but more often in pairs, taken with their 
source, makes it seem more than likely they were pneumococci, but 
Eberth thought they were only varieties of diphtheria or pyemia 
micrococci. Matray, 869 " 70 in the same year, gave the name Pneu- 
moniekokken to the cocci which he found in the sputum of pneu- 
monia patients and in normal sputum as well. Had animal inocu- 
lation been tried by either Eberth or Matray their find would have 
preceded the real discovery by Pasteur and Sternberg by over a 
year; but it was not tried. 

In 1881, these two authors, Pasteur and Sternberg, independ- 
ently inoculating rabbits, the former with the saliva of a child 



HISTORY OF PNEUMOCOCCUS: 1875-1890 3 

dead from rabies, the latter with normal saliva, isolated Pneumo- 
coccus for the first time through animal passage. Pasteur, con- 
trary to the subsequent opinions of others, thought that he had 
discovered a new disease.* Sternberg reported that the fatal septi 
cemia which normal saliva produced in rabbits was due to an or- 
ganism that later, upon microscopic examination, appeared to 
agree exactly with the description given by Pasteur. 

Pasteur 1065 " 6 passed his virus from infected to normal rabbits, 
producing infection each time, recovered the organism from the 
blood by cultivation in bouillon and other media, and with these 
cultures induced a similar fatal septicemia in other rabbits. On 
microscopic examination he found the bacteria to be very short 
rods, somewhat depressed in the center, with a visible aureola due, 
as he thought, to mucin. 

Sternberg 1316 " 8 ascertained that the ability to incite fatal rab- 
bit septicemia was a peculiarity of the saliva of some normal in- 
dividuals but not of others, and that normal blood, putrid urine, 
liquid feces, and bouillon undergoing spontaneous putrefaction 
failed to produce a similar infection. In a later study appearing 
after the publication of Pasteur's paper, using aniline violet as a 
stain and making a permanent record by photomicrographs, 
Sternberg confirmed Pasteur's and his own findings. Here the dates 
are worth noting. Pasteur announced his discovery before the 
French Academy of Sciences in December, 1880, the report ap- 
pearing in the Academy's Comptes Rendus in January, 1881. 
Sternberg began his experiments in the summer of 1880, and made 
his report which was first published in April, 1881. 

There then arose a misconception of Pasteur's claims which was 
to persist for a long time. By his fellow countryman, Colin, 269 he 
was gratuitously credited with the claim that he had isolated the 
microbe of rabies. Pasteur emphatically replied, J'ignore absolu- 
ment les relations de cette nouvelle maladie avec la rage. Pasteur 

* The original communication was presented by Pasteur, but the work was 
done with the collaboration of Chamberland and Roux. 



4 BIOLOGY OF PNEUMOCOCCUS 

also stated that this new disease was not a septicemia, and this mis- 
take undoubtedly contributed to the confusion of ideas. Disturbed 
by this false imputation, Pasteur, in March, 1881, in a letter to 
Parrot 1064 who had, like Sternberg, produced a fatal infection in 
rabbits with normal saliva, again stated positively that his or- 
ganism bore no relation to rabies. Pasteur, by the way, showed his 
great perspicacity in a paragraph which was included in that let- 
ter. It bears repeating: J'y vois, pour ma part, un symptome nou- 
veaw de grand avenir pour la connaissance etiologique des maladies 
dont la cause doit etre attribuee a la presence et au developpement 
d'organismes micro sco piques. Again, in a communication read at 
the May 31st session of the Academie de Medecine, Pasteur 
sharply protested against the claims ascribed to him. 

In this connection, the originals of Pasteur's and of Sternberg's 
later papers (1885 and 1887 ) 1319 " 20 are well-worth reading. In 
these days of petty priority claims it is refreshing to read of these 
two gentlemen of the eighteen-eighties, of whom one, Pasteur, 
frankly acknowledged his ignorance, while the other granted prece- 
dence and magnanimously named the organism after Pasteur. 
Sternberg's statement (1885) ran as follows: 

In attaching to this micrococcus the name of the illustrious French 
chemist I have no desire to perpetuate the memory of the mistake he 
made in supposing for a time that it was the germ of hydrophobia. Hav* 
ing found that this was a mistake, he did not fail to correct it. . . . It 
is easy to make mistakes in this field of investigation ; easier, perhaps, 
than to acknowledge them.* And believing, as I do, in human fallibility, 
I have no hesitation in questioning the conclusions of the most illus- 
trious workers in the field of microbiology, if they are in conflict with 
my own observations. On the other hand, if, upon fuller investigation, I 
am convinced that I have been mistaken in regard to this or any other 
question, I shall feel no hesitation in following the example of Pasteur 
in making a public announcement of my error. 

Here is material for a Hippocratic oath for the biologist or any 

other fellow of science. 

* Sternberg was prominent among those who had given an entirely errone- 
ous interpretation to Pasteur's claims, and this statement, gracious though it 
was, served to foster the unfortunate misconception. 



HISTORY OF PNEUMOCOCCUS: 1875-1890 5 

Vulpian, 1453 at the March (1881) meeting of the French Acad- 
emy, said that he regretted that he had not been present at a for- 
mer session when the letter from Pasteur to Parrot had been read, 
because he also might have reported that he had caused the death 
of a rabbit by the subcutaneous injection of normal saliva, while 
the blood of that animal killed another rabbit within the space of 
two days. The blood of the animals contained a number of "mi- 
crobes," the majority having the same characteristics as those de- 
scribed by Pasteur. Here was the link between the work of Pasteur 
and that of Sternberg, but Vulpian, besides failing to appreciate 
the connection, was tardy in his announcement. Claxton (1882), 237 
too, found normal human saliva infectious for rabbits. Believing in 
Pasteur's idea, he however confirmed Sternberg's results, with the 
added observation that human saliva varied in its infecting power, 
that of individuals from such tropical countries as Cuba and Bra- 
zil being extremely virulent. 

In 1881, Osier 1039 spoke of finding "micrococcus balls" in four 
cases of endocarditis complicating pneumonia. He was not willing 
to assert they were the cause of the disease, but called attention to 
the frequency with which this condition and meningitis accompany 
pneumonia. Osier's comment recalls the earlier description by 
Koester (1878) 736 of "bullet-like masses of micrococci" in embolic 
endocarditis, which may or may not have been pneumococci. 

The year 1881 was an eventful one. If one examines the photo- 
micrographs accompanying Robert Koch's 735 paper on pathogenic 
organisms, there is little difficulty in identifying these lance-shaped 
diplococci, but to Koch they were that and nothing more. Poin- 
care 1102 was nearer the truth when he spoke of finding "prodigious 
numbers of little spheres of double contour" in pneumonic lung tis- 
sue, but he lacked the support of positive animal experiments to 
justify a claim that these organisms were responsible for pneu- 
monia. 

Shortly after the preliminary announcements of Pasteur and of 
Sternberg, Friedlander ( 1882) 486 presented a communication which 
takes high rank in the history of Pneumococcus. He reported that 



6 BIOLOGY OF PNEUMOCOCCUS 

in September, 1881, in the fibrinous exudate and in hardened sec- 
tions of lung and pleural tissue, stained by the Weigert-Koch 
method, from eight cases of acute pneumonia and in the fluid with- 
drawn from living pneumonia patients by lung puncture, he had 
observed spherical and ellipsoidal cocci, occurring mostly in pairs, 
and sometimes in longer chains made up of diplococci. 

In 1882, Giinther, 579 at a meeting of the Verein fiir tnnere Medi- 
zin in Berlin, had reported that the microscopic examination of 
stained preparations of purulent, bloody matter from lung punc- 
ture showed numerous diplococci. He gave a demonstration of his 
preparation and exhibited a drawing which caused Fraenkel to re- 
mark that the cocci seemed to be surrounded by an unstained rim. 
Giinther replied that the rim was really a hull. Early in the same 
year, Colomiatti, 271 who was familiar only with the work of Klebs, 
reported finding Monas pulmonale in a growth on the heart valve 
and in the subarachnoid fluid in a case of croupous pneumonia 
complicated with endocarditis and meningitis. 

In 1883,* Friedlander 4878 announced that he had found the same 
micrococci in the alveolar exudate in all but a few of fifty cases 
of pneumonia. Stained by the method of his colleague, Gram, 
these cocci took an intense color, which was not extracted by treat- 
ment with alcohol, and exhibited a well-defined capsule. The cap- 
sule, which a short time before had been observed by Giinther, 
could be clearly demonstrated by fixing the preparations with 
acetic or mineral acids and staining with gentian violet, fuchsin, 
Bismarck brown, or methylene blue. The capsular substance was 
insoluble in alcohol, ether, or chloroform, and was best shown by 
preliminary staining with aniline gentian violet, a short exposure 
to alcohol, and counterstaining with eosin. The contour was 
brought out by osmic acid but there was no blackening of the cap- 
sule or cell. Friedlander observed that the capsules were most char- 
acteristic at the highest stage of the cells' development, and con- 

* A greater part of this work was done with the collaboration of Frobenius, 
who, because he did not remain until the completion of the study, declined the 
inclusion of his name in the authorship of the paper ! 



HISTORY OF PNEUMOCOCCUS: 1875-1890 7 

sidered them not as a passive precipitate, but as a product of the 
vital action of the coccus. Applying several microchemical tests to 
the capsular material, he felt justified in concluding that it con- 
sisted of mucin or an allied substance. When one remembers that 
mucin is a protein-carbohydrate complex in which the saccharide 
yields on hydrolysis two glucosamin, one acetic acid, and two 
hexose molecules, one appreciates the value of this observation in 
anticipating the discovery of the soluble specific substance or cap- 
sular polysaccharide by Heidelberger and Avery. Friedlander cul- 
tivated these organisms on Koch's coagulated blood serum, upon 
which they grew as round and elliptical cocci. His descriptions of 
the colonies on this medium corresponded with the appearance of 
the colonies of typical pneumococci. He also said that he had car- 
ried the cultures through eight transfers in meat-infusion peptone 
gelatin. In this medium the coccus lost its capsule and grew in 
"nail-form" colonies. 

When one-half to one cubic centimeter of aqueous suspensions of 
the gelatin cultures was injected into the lungs of rabbits no in- 
fection resulted. Guinea pigs inoculated in this way were more sus- 
ceptible, six of nine becoming infected. Mice were far more sus- 
ceptible, all dying of infection. From the pleural and lung exudate 
of the infected animals Friedlander isolated typical diplococci. In 
four of twelve mice he succeeded in producing infection by inhala- 
tion. One of four dogs succumbed to the injection of cultures into 
the lung, and this animal at necropsy showed red and gray hepa- 
tization of the lung. From these areas, from the blood, and from 
the right pleural cavity, Friedlander recovered typical organisms. 
He noted variations in the capsule of the organisms depending 
upon their propagation in mice, guinea pigs, dogs, and man. 

At that time, Friedlander was unable to demonstrate cocci in the 
blood of pneumonia patients, but in 1884 489 " 90 reported that he 
had cultivated typical micrococci from the blood of one of six pa- 
tients. The cocci were encapsulated and virulent for mice, but by 
the method tested were avirulent for rabbits. It was in his first 



8 BIOLOGY OF PNEUMOCOCCUS 

communication that, after giving credit to Klebs for the first ref- 
erence to Pneumococcus and to Eberth and Koch for previous re- 
ports, Friedlander gave the name Pneumoniemikrokokken to the 
organisms. He believed that the capsule and the nail-form colony 
were not distinctive diagnostic characters, but that the whole cycle 
of phenomena must be observed, namely, the effect on animals and 
recultivation of the cocci from the artificially infected animals. He 
agreed that it was impossible to obtain this complete series of 
events in all cases of pneumonia, and suggested that there might be 
several forms of pneumonia, one of which was caused by the Pneu- 
moniemikrokokken, or that the organisms while present at one 
stage of the disease were either not present or dead at other stages. 
He inclined to the first viewpoint. 

Salvioli and Zaslein (1883) 1217 examined the sputum, canthari- 
des blister fluid, and the blood of pneumonia patients. Here the 
same cocci, less frequent in the sputum than in the other fluids, 
could be cultivated in broth and in Pasteur's solution and ren- 
dered visible with Bismarck brown and methyl violet. 

In 1883, Strassmann 1343 also found diplococci in pneumonic 
sputum. 

In the same year, before the Berlin Medical Society, von Ley- 
den demonstrated in blood and exudate aspirated from the hepa- 
tized lung of a pneumonia patient diplococci, largely oval in form, 
occasionally in longer chains. He added that similar cocci could be 
seen, without staining, in small masses scraped from the affected 
lung at necropsy. He surmised that Klebs had seen the same or- 
ganisms. 

Considering the meagerness of information in his time (1882), 
Bozzolo 145 contributed some sound ideas. He concluded from his 
studies that pleurisy, pericarditis, endocarditis, and cerebrospinal 
meningitis were frequently associated with lobar pneumonia ; that 
this association, whether the disease occurred in epidemic or spo- 
radic form, was caused by the action of a single infecting agent ; 




Courtesy of the Journal of the American Medical Association 



GEORGE MILLER STERNBERG 1838-1915 






Photograph by Keystone View Co. 

ALBERT FRAENKEL 1848-I916 
By Alexander Oppler, Stadtisches Krankerihaus, Berlin 



HISTORY OF PNEUMOCOCCUS: 1875-1890 9 

and that the agent, as demonstrated by sufficient scientific facts, 
could be said to be a species of Schyzomycetes. 

Ziehl, 1574 familiar with the work of Klebs, Eberth, Friedlander, 
and von Leyden, sought bacteria in the sputum of two pneumonia 
patients. He saw ellipsoidal cocci in the early stages of the disease, 
and the same organism mixed with many other bacterial forms in 
the later stages. Peiper 1076 reported the presence of large numbers 
of diplococci in lung-puncture fluid. That primary sporadic cere- 
brospinal meningitis could be caused by Pneumococcus was first 
discovered by von Leyden 810 but, because the oval diplococci ap- 
peared to be slightly larger than those found in pneumonia, he was 
in doubt as to their true nature. 

Late in 1883, Talamon, 1377 although lacking conclusive experi- 
mental data, felt justified in stating that lobar, fibrinous pneu- 
monia was an infectious disease produced by a specific microbe 
with a characteristic form, and he named the organism, Coccus 
lanceole de la pneumonic He found this coccus in the pulmonary 
exudate taken by lung puncture, and once in the blood during 
life. Since only a fluid medium was used, the cultures were seldom 
pure. Talamon failed to infect guinea pigs or dogs with this mixed 
growth, but sixteen out of twenty rabbits succumbed to inocula- 
tion, although none developed pleuritis, pericarditis, or pneu- 
monia. Notwithstanding the fact that his experimental results did 
not fulfill all the requirements of Koch's law, the claim was cor- 
rect, and the statement that pneumonia is a localized disease, prin- 
cipally pulmonary, and susceptible of becoming generalized in 
other organs, was true.* 

During the year 1884, interest in Pneumococcus spread and 
fresh information came from various sources. Gram 545 himself gave 
the details of the indispensable stain which now bears his name, 
and described how the capsule first took the dye and then gave it 

"Mendelsohn (1884)892 gave an excellent review of the current ideas on the 
epidemiology and infectiousness of pneumonia. 



10 BIOLOGY OF PNEUMOCOCCUS 

up to alcohol, while the coccus retained the violet color. Then Em- 
merich, 355 after hunting bacteria in air, water, dust, and dirt, re- 
covered encapsulated diplococci from the filling between the floors 
of a prison in Augsburg, notorious for over twenty-five years as a 
breeding place of pneumonia. He cultivated the cocci on meat- 
infusion peptone gelatin, separated them from accompanying bac- 
teria, injected the purified culture into rabbits with indifferent suc- 
cess, but obtained a higher fatality rate when he used mice. He 
identified the coccus as that described by Friedlander, and gave 
to the report the title, Die Auffindung von Pneumoniecoccen. . . . 
Emmerich's discovery, confirmed several decades later by the work 
of Stillman, 1326 furnished one of the first epidemiological clues to 
the origin of some cases of pneumonia. 

Somewhat analogous to Emmerich's discovery of pneumococci in 
the floor filth of a pneumonia-infected prison was the apparent 
success of Pawlowsky (1885) 1072 in isolating, on solid media, Pneu- 
mococcus-like organisms from the air of various rooms. Influenced 
by the teaching of Friedlander, Pawlowsky, because his cocci were 
slightly smaller (they were also associated with larger cocci) and 
because they were pathogenic for rabbits, was not sure of his 
ground. The infectiousness of these cocci for rabbits, guinea pigs, 
and dogs indicates that among the bacteria described by Paw- 
lowsky there were pneumococci. Cornil and Babes 278 added a note 
to the effect that they had found lancet-shaped bacteria in the ton- 
sils and from the endocardium of pneumonia patients, but they 
made no cultures. 

Exudates from the pleura and pericardium of two pneumonia 
patients yielded, in Salvioli's 1215 hands, encapsulated cocci which, 
while fatally infective for guinea pigs, rabbits, and dogs, failed to 
cause the typical lesions of pneumonia. Thinking that he had over- 
whelmed the animals with the virulence of the fluids injected, he in- 
troduced the infectious material intratracheally into four guinea 
pigs, two of which died. Platonow, 1095 in a detailed and critical re- 
view of the existing literature, concluded from the evidence and his 



HISTORY OF PNEUMOCOCCUS: 1875-1890 11 

own experimental experience that neither the capsule nor the nail- 
form colony was a differential character and that bacteriological 
examination of the blood of pneumonia patients was not of diag- 
nostic value. From Platonow's notes it would seem that he used 
mixed cultures of bacilli and cocci, so his doubts were well founded. 

In April of that year (1884), Fraenkel 466 presented the first of 
a series of studies that were to make the name Fraenkel and Pneu- 
mococcus almost inseparable. In a discussion of the cause of pneu- 
monia before the Kongress filr innere Medizin in Berlin, Fraenkel 
displayed a human trait that at least enlivens the sober literature 
if it does not always bring the desired personal reward. Fraenkel 
complained that he should receive some of the credit for the dis- 
covery of Pneumococcus. He had begun experiments six months 
before Friedlander's report was made public, and had only delayed 
the announcement because his results differed from Friedlander's 
and because he wished to recheck them ! He had scored on his fel- 
low countryman by infecting rabbits with the coccus where Fried- 
liinder failed, but his further observations had not been so positive. 
There was a difference in the pathogenicity of some of the mate- 
rials which he used; colony appearance and capsule formation 
were not constant characters and, moreover, other bacterial spe- 
cies had capsules. Apparently unaware of Sternberg's experiments, 
Fraenkel 468 had missed the meaning of his success in producing a 
fatal septicemia in rabbits with normal sputum and the subsequent 
recovery by cultivation on coagulated blood serum of encapsulated 
diplococci from the blood of the animals. He had hesitated to draw 
any comparison between the diplococci from pneumonic material 
and his "sputum septicemia coccus." Likewise, he had ventured no 
outright statement that his and Friedlander's organisms were sepa- 
rate species ; in fact, he gave the impression that they were differ- 
ent forms of the same organism. Friedlander, in the discussion of 
Fraenkel's paper, suggested that there might be several organisms 
causing pneumonia, so he, for the while, had the last word. 

Here began a controversy, fomented principally by Fraenkel, 



12 BIOLOGY OF PNEUMOCOCCUS 

despite the conciliatory attitude of Friedlander, which led to con- 
fused conceptions which endure to the present day and which can 
never be clarified. Fraenkel, in a paper published in 1885, 407 virtu- 
ally acknowledged that the coccus described by Friedlander and 
the one described by himself were identical, but complained that he 
saw it first. It has long been the verdict that the organism de- 
scribed by Friedlander in 1882 and 1883 was not Pneuniococcus 
but the bacillus which later came to bear his name. This verdict 
would deprive Friedlander of any credit for the original isolation 
of Pneumococcus from lobar pneumonia and bestow it on Fraenkel 
who, without it, still has the honor of giving the first complete de- 
scriptions of Pneumococcus and whose studies proved for the first 
time the etiological relationship between this coccus and lobar 
pneumonia in man. 

The facts given in Friedlander's first and second papers admit 
of more than one interpretation. The organism which Friedlander 
first described was an elliptical, or round, encapsulated coccus oc- 
curring principally in pairs. It was isolated from nearly all of 
fifty cases of acute pneumonia and was Gram-positive. It need 
scarcely be pointed out that these are characters of Pneumococcus 
and not of B. friedlanderi* The culture was exquisitely virulent 
for mice. However, the lack of pathogenicity for rabbits, its abil- 
ity to grow on gelatin at room temperature with the formation of 
nail-like colonies are not characteristic of typical pneumococci. 
Perhaps Friedlander, in the face of strong opposition, weakened 
his position by acknowledging that the cycle of isolation of the 
coccus from man, the production of infection in animals, and its 
subsequent recovery could not be accomplished in every case of 
pneumonia. Possibly another factor tending to detract from the 
true import of his discovery may have been his suggestion that 
there might be different forms of Pneumococcus, or that pneu- 

* Inasmuch as the earlier name, Bacillus friedlanderi, occurs in the great 
majority of the original publications reviewed, it, rather than the newer name, 
Klebsiella pneumoniae, will be used in this text. 



HISTORY OF PNEUMOCOCCUS: 1875-1890 13 

monia might be caused by other organisms ; ideas that we now 
know are true but which were scouted at the time. A careful read- 
ing of Friedlander's original communications makes it difficult to 
escape the conviction that he, in 1881, saw in the secretions and 
tissues of pneumonia patients the organism we know as Pneumo- 
coccus and, in the next two years, accomplished its isolation and 
cultivation. It seems likely, however, that Friedlander isolated at 
the same time the bacillus which later came to bear his name. 

One seems justified in assuming as a cause of the divergent opin- 
ions which have so long persisted in the literature, the statement in 
Friedlander's communication of 1886 492 that the micrococcus so 
thoroughly studied by Fraenkel was the coccus he had originally 
described, but that the organism upon which he was then reporting 
was neither a coccus nor a bacillus* but a bacterium which he 
called Kapselbacterium. In addition to short elements, it existed 
in rod-like and thread-like forms and, furthermore, it was found 
only in a minority of the pneumonia cases studied — both charac- 
ters of the Friedlander bacillus. Therefore, in view of all the evi- 
dence, and with due deference to the opinion of distinguished au- 
thorities, it would seem that the credit for first indicating that a 
diplococcus — Diplococcus pneumoniae — might be the cause of 
pneumonia should be given to Friedlander. To him, also, should go 
the honor of discovering another etiological agent of pneumonia, 
Bacillus friedlanderi. 

Afanassiew, 4 in 1884, added suggestive, if somewhat doubtful, 
information to the question. He, too, obtained ovoid cocci from six 
cases of pneumonia, but the cultures were not pure and this fact 
led to uncertainty. Klein, 719 on the contrary, apparently succeeded 
not only in growing pure cultures of Pneumococcus and in infect- 
ing mice and rabbits with them, but in recovering the organisms 
from the test animals and in transmitting the infection in series to 

* It should be borne in mind that, at the time, the criterion for judging 
whether an organism was a bacillus or a bacterium was the presence of mo- 
tility in the former and its absence in the latter. 



14 BIOLOGY OF PNEUMOCOCCUS 

other rabbits and mice. The infection was a septicemia and never a 
localized process as claimed by Friedlander. Klein had no hesita- 
tion in using the name Pneumokokkus, in the title of his paper. 

Maguire (1884), 855 in England, presented before the British 
Medical Association three preparations to illustrate the micrococ- 
cus of pneumonia. One was a section of a pneumonic lung, another 
a section of kidney from a case of pneumonia, and the third, spu- 
tum from a similar patient. Employing both methylene blue and 
the Gram stain, he found capsules on the cocci, although they were 
not always present. Maguire exhibited the specimens as illustrat- 
ing Friedlander's micrococcus but, lacking cultural or inoculation 
experience, showed caution in saying that the question of the role 
of Pneumococcus must remain in abeyance until more data were 
forthcoming. 

In the same year, Foa and Rattone, 464 working with a pure cul- 
ture of Friedlander's "Pneumococcus" obtained from his colleague, 
Frobenius, duplicated Friedlander's observations even to the re- 
sistance of rabbits to its invasion. They confirmed the results with 
cultures isolated from the lung of a pneumonia patient, and were 
the first to succeed in producing meningeal infection in guinea 
pigs by inhalation, recovering "capsule-cocci" from the exudate 
on the pia mater.* 

By 1885, Sternberg 1319 was ready to state that the pneumonia- 
coccus of Friedlander, the Microbe septichnique du saliva of Pas- 
teur, and the organism he himself had isolated from normal saliva 
were not only the same but were probably the cause of pneumonia.f 
His conclusion bears quoting: "It seems extremely probable that 
this micrococcus is concerned in the etiology of croupous pneu- 
monia, and that the infectious nature of this disease is due to its 
presence in the fibrinous exudate into the pulmonary alveoli." Its 

* Jiirgensen699 reviewed the literature up to his time (1884) and concluded, 
"True pneumonia is an infectious disease, which principally but not exclu- 
sively, affects the lungs." 

t Sternberg, after seeing the Friedlander culture in Koch's laboratory in 
1885, changed his mind, and in 18871320 sa id he was in error in thinking that 
this and his saliva-coccus were identical. He then tentatively changed the name 
to Micrococcus pneumoniae crouposae. 



HISTORY OF PNEUMOCOCCUS: 1875-1890 15 

presence in normal human saliva seemed to Sternberg to indicate 
that some other factor was necessary for the development of an at- 
tack of pneumonia. It was possible that some condition, such as 
"alcoholism, sewer-gas poisoning, crowd-poisoning or any other 
depressing agency" might render the individual vulnerable, while a 
"reflex vaso-motor paralysis, induced by cold, might affect a single 
lobe of the lung." It was at this time that Sternberg graciously 
gave the name Micrococcus Pasteuri to the coccus. 

Fraenkel (1886), 468 now reassured by his experiments but still 
conservative, issued another note, preliminary to a more positive 
statement to appear later, to the effect that diplococci were some- 
times, but not always, found in normal human saliva, more often in 
the saliva of a sick person, and still more frequently in the rusty 
sputum of pneumonia patients. He called the disease produced by 
this organism in rabbits and mice, "sputum septicemia." The or- 
ganisms failed to grow on gelatin at room temperature, but on 
congealed blood serum or on agar at body-heat developed charac- 
teristic veil-like or dewdrop-like colonies. A similar coccus was 
present in two cases of empyema following pneumonia, but in some 
cases of empyema other organisms were to be found in the pus. 
Fraenkel then went so far as to say that these facts would make it 
seem highly probable that the microbe of sputum septicemia and 
Pneumococcus were identical. 

In the following year (1886), more positive in his convictions, 
Fraenkel 469 was ready to grant that his coccus — that of sputum 
septicemia — the sputum coccus of Pasteur and that of Sternberg, 
as well as the organisms described by Talamon and by Salvioli, 
were identical, and then to state definitely that this coccus was the 
cause of true fibrinous pneumonia. With new assurance Fraenkel 
became somewhat caustic in his comment about Friedlander's 
claims and flatly avowed that his organism and that of Friedlander 
were not the same. He then named it Pneumoniemikrococcus, call- 
ing it also Pneumococcus. Here, more so than previously, Fraen- 
kel's experiments are impressive for their care and thoroughness. 



16 BIOLOGY OF PNEUMOCOCCUS 

He grew the cocci on liquid and solid media, studied the most 
favorable reaction for growth, and found that while the organ- 
isms retained their virulence on coagulated blood serum or meat- 
infusion agar, they lost it rapidly in broth because of its changed 
reaction during incubation. Young cultures were highly patho- 
genic for mice and rabbits, less so for guinea pigs, and avirulent 
for dogs, pigeons, and chickens. 

Fraenkel was able to isolate the diplococcus from all cases of 
true fibrinous pneumonia, grew it in pure culture, produced a fatal 
septicemia in susceptible animals, again recovered from them the 
organism in a pure state, and then further transmitted the infec- 
tion to other test animals. His results therefore met all the require- 
ments of Koch's law, and it would seem that Fraenkel was the first 
to establish the fact beyond a reasonable doubt that Pneumococcus 
was the causative agent in lobar pneumonia. 

Fraenkel 468 made another important and original contribution 
when he reported that rabbits, recovering from a subcutaneous in- 
fection of the ear, resisted a subsequent inoculation with the same 
coccus. Although Foa and Bordoni-Uffreduzzi had independently 
discovered the same phenomenon (1884), this is probably the first 
controlled observation that pneumococcal infection may induce 
immunity. 

Fraenkel also found Pneumococcus in the exudates in the pia 
mater from a case of meningitis accompanying pneumonia, but was 
unable to decide which lesion was primary. Senger (1886) 1255 had 
seen these diplococci in amazing numbers in fluid from the sub- 
arachnoid and ventricular spaces in similar cases and, in addition, 
in the lesions of endocarditis, pericarditis, pleuritis, and nephritis 
of pneumonia patients. He was, of course, correct in supposing 
that these and other metastases might be expected to arise from a 
pulmonary lesion. Netter, 962 " 3 ' 965 and also Lancereaux and Be- 
sancon, 778 reported the same findings in two corresponding cases of 
meningitis, pericarditis, and endocarditis. Lebashoff, 795 in Russia, 
at this time announced similar observations on the occurrence of 




Figure 1 



Figure 2 




Figure 3 



Figure 4 



After Sternberg 1 



MICROCOCCUS PASTEURI, 1 885 

Figure 1. Micrococcus Pasteuri from blood of rabbit inoculated sub- 
cutaneously with normal human saliva (Dr. S.). Stained by the method 
recommended by Friedlander. Magnified 1000 diameters. Figure 2. 
Micrococcus Pasteuri from blood of rabbit inoculated subcutaneously 
with fresh pneumonic sputum from a patient in the seventh day of the 
disease. Same staining and amplification as Figure 1. Figure 3. Sur- 
face culture of M. Pasteuri, showing development of long chains. Same 
staining and amplification. Figure 4. Surface culture of M. Pasteuri 
from blood of rabbit injected with pneumonic sputum, showing the so- 
called "capsule" of Friedlander. Same staining and amplification. 



HISTORY OF PNEUMOCOCCUS: 1875-1890 17 

these diplococci in other organs as well as in lung tissue but, evi- 
dently being ignorant of Fraenkel's work, he described the organ- 
isms as Friedlander's cocci. 

In France, Gamaleia (1886), 498 after isolating, cultivating, and 
inoculating into several different species of animals, cocci obtained 
from lungs and fluids from meningitis and endocarditis complicat- 
ing pneumonia, announced that the encapsulated lancet-form dip- 
lococcus derived from these sources was the same as the organism 
originally described by Pasteur. As a compliment to his chief, but 
deceived by the occasional chain formation, Gamaleia named it 
Streptococcus lanceolatus Pasteuri. In offering an explanation for 
the resistance of man to its invasion, he drew attention — an origi- 
nal observation — to the possible part played by leucocytes in the 
body's defense. 

Pneumococcal infection of the kidneys was first reported by 
Nauwerck 944 " 6 in 1886. Among 550 cases of croupous pneumonia 
there were thirteen complicated with acute nephritis and in these 
instances he found in the kidneys cocci which he said were identical 
with those described by Friedlander. Nauwerck stated that the in- 
fection was specific and was caused by "pneumonia cocci" carried 
to the kidney by the blood. 

At this time the differentiation began between Fraenkel's Pneu- 
mococcus and the bacillus of Friedlander. Weichselbaum, and Foa, 
and Bordoni-Uffreduzzi sharply distinguished between the two» or- 
ganisms, although it may be again emphasized that Friedlander's 
first descriptions were those of a diplococcus and not of a bacillus. 
Some of the credit usually given to Fraenkel's contributions may 
be granted to Foa and Bordoni-Uffreduzzi 460 whose work was done 
independently of Fraenkel's and was actually made public some- 
what earlier. 

A full confirmation of Fraenkel's work, if not of his conclusions, 
came from Weichselbaum. 1497 ' 8 Beginning his studies shortly after 
Friedlander's first announcements, Weichselbaum covered the 
ground gone over by the earlier investigator, found the diplococci 



18 BIOLOGY OF PNEUMOCOCCUS 

in a percentage of cases sufficiently high to promise diagnostic 
value, and gave clear and detailed descriptions of their isolation, 
cultivation, and animal pathogenicity. He added the gums, tonsils, 
accessory sinuses, cerebrospinal fluid, and joints as sites of pneu- 
mococcal infection. The organism differed, as in Fraenkel's experi- 
ence, from Friedlander's in that it failed to grow on gelatin, while 
the author emphatically differed from Fraenkel in the belief that 
this coccus was the sole cause of pneumonia. 

Weichselbaum (1886) 1498 " 1500 separated pneumonic affections 
into lobar, croupous, lobular, and hypostatic pneumonias, fixed on 
Pneumococcus the guilt of causing the lobar type, and on strepto- 
cocci, staphylococci, and a bacillus (Bacillus pneumoniae — prob- 
ably the Friedlander bacillus) that of producing the other types. 
He presented new data concerning the morphology and growth 
characters of Pneumococcus ; from the infected lung tracked the 
organism through the lymphatics to the cerebral ventricles ; and 
suggested the blood stream as a further avenue of its spread. To 
the organism Weichselbaum gave the name Diplococcus pneu- 
moniae. In the next year (1887), Weichselbaum repeated Stern- 
berg's work and, in addition, by the subcutaneous injection of 
dried spleens from animals succumbing to the diplococcal infec- 
tion, and of post-pneumonic pleural exudate containing attenuated 
pneumococci, immunized mice and rabbits. He, like Fraenkel, called 
the organism Pneumococcus. 

In the same year, confirmation of the diversity of organisms in- 
volved in pneumonia came from Wolf, 1529 while Meyer 895 offered 
further proof of the presence of pneumococci in the lungs, heart, 
and cerebrospinal exudates in infections secondary to pneumonia. 
Netter, 965 finding these same cocci in the nasal fossae, sinuses, and 
tympanic cavity, then made the original observation that a local 
process, such as pneumococcal meningitis, could arise without a 
contributing lung infection. 

Sternberg, not yet entirely convinced that the lanceolate diplo- 
coccus was the true cause of pneumonia, but hoping for a better 



HISTORY OF PNEUMOCOCCUS: 1875-1890 19 

understanding of his work by the German bacteriologists, pre- 
sented his views (1887) in a paper 1320 in the Deutsche medizinische 
Wochenschrift. He first admitted his original error in thinking 
that his saliva-coccus and the organism of Friedlander were the 
same ; then went on to say that if this diplococcus could finally be 
proved to be the cause of true pneumonia, it should be called Mi- 
crococcus pneumoniae crouposae, but until that time the name he 
originally proposed should stand. This statement aroused Fraen- 
kel, 471 and a month later, again feeling that he had been slighted, 
he took issue in print with the American, and made another plea 
for priority. Fraenkel characterized Sternberg's work as incom- 
plete, made capital out of the confusion of the Sternberg coccus 
with Friedlander's bacterium, and disposed of Sternberg's objec- 
tion to Fraenkel's name, Pneumoniemikrococcus. 

Foa and Bordoni-Uffreduzzi 460 " 2 put in a bid for credit as be- 
ing the first to report (1886) the discovery of the organism to 
which they gave the name Diplococcus lanceolatus in a case of pri- 
mary cerebrospinal meningitis. They also reported the isolation of 
the organism from every case in an epidemic of the disease,* as well 
as from cases of polyarthritis and from the blood of the placenta 
in a patient aborting during pneumonia. Ortmann, 1037 too, isolated 
pneumococci from meningeal infections, and in so doing felt that 
he should be acclaimed as the first to obtain "capsule-cocci" on 
artificial media. Infection of the parotid glands was another mani- 
festation of the invasiveness of Pneumococcus, two instances in 
which parotitis occurred being reported by Testi, 1388 " 90 who also 
noted subcutaneous abscesses as a further complication of pneu- 
monia. 

More important than the priority claim of Foa and Bordoni- 
Uffreduzzi, however, was their achievement in immunizing rabbits 
by subcutaneous injection at three or four-day intervals, first with 
attenuated material, then with cultures of increasing virulence. 
The animals thus treated became resistant to inoculation with 

* It is probable that the epidemic was one of meningococcal meningitis. 



20 BIOLOGY OF PNEUMOCOCCUS 

virulent cultures or infected blood. Encouraged by these results, 
the authors applied the procedure to human beings but without 
success. Incidentally, Foa and his colleague were among the first to 
maintain virulence of the cocci by preserving the cultures in in- 
fected blood in the cold and the dark. So also, Biondi, 117 in experi- 
ments with Bacillus salivarius septicus (the Fraenkel or Sternberg 
diplococcus in spite of the name), by chance noticed that rabbits 
recovering from inoculation with weakened cultures became im- 
mune. Biondi looked upon such attenuated cultures as true vac- 
cines. Netter 971 accomplished the same result with the dried spleens 
of animals dying from pneumococcal infection and with "old" 
pleural exudate containing this organism. 

Zaufal (1888) 1568 " 9 contributed otitis media to the list of pri- 
mary infections due to Fraenkel's diplococcus, and sharply differ- 
entiated this coccus and the Friedlander bacillus, which was also 
found to be responsible for a similar condition. Then Gamaleia, 498 
harking back to the Pasteur organism, told of its constant pres- 
ence in fibrinous pneumonia, and insisted that the Friedlander ba- 
cillus was a saprophyte. 

In 1889, Foa and Bordoni-Uffreduzzi 461 introduced a confusing 
note with the isolation of a monococcus from some mild cases of 
fibrinous pneumonia. They suggested the possibility of there being 
two types of organisms, but the descriptions are too incomplete to 
warrant a decision. 

The names of Fraenkel and Weichselbaum had now become 
practically hyphenated when applied to Pneumococcus. Arusta- 
mow (1889) 24 found this organism in every one of fifty cases of 
pneumonia, but failed in the examination of the saliva of fifteen 
normal persons and the sputum from a like number of bronchitis 
patients. Gabbi 497 isolated in pure culture the Virus pneumonico 
(Microbio capsulato del Fraenkel) from a peritonsillar abscess 
without pneumonia ; while Monti, 905 after finding Pneumococcus in 
an arthritic joint of the hand, induced a localized infection experi- 
mentally in a rabbit with the strain he isolated from the joint. 



HISTORY OF PNEUMOCOCCUS: 1890-1900 21 

The decade of the eighties saw the first important chapter in the 
history of Pneumococcus written, edited, and given to the medical 
world. Its causative association with pneumonia, meningitis, and 
certain localized infections was accepted as established despite 
some confusion concerning its various identities. Fraenkel, and not 
Friedlander, was now looked upon as its true sponsor. The early 
and necessarily crude and incomplete immunization experiments 
were to encourage a closer study of this phase of the activity of 
Pneumococcus. The next ten years were to be less fruitful, but here 
and there facts were disclosed which, with a better understanding, 
were to take on a new significance. 

1890-1900 

In the second decade, Foa and Carbone 463 used soluble products 
of Pneumococcus to stimulate the immune response in rabbits, and 
sought by chemical means to refine and concentrate the antigenic 
principle — believed to be a poison or toxin — elaborated during 
pneumococcal growth. The authors went no further than to say 
that while the refined substance, which had been precipitated by 
ammonium sulfate and refined by dialysis, failed to kill the animal, 
it produced marked physiological changes — a statement which is 
scarcely descriptive of any specific action. 

The Klemperers (1891) 723 " 6 might justly be looked upon as the 
forefathers of antipneumococcic serum therapy. They immunized 
rabbits with sputum obtained from pneumonia patients after re- 
covery, with purulent, but bacteria-free, pleural exudate, with 
heated glycerol extracts of pneumococci, and with heated whole 
and filtered broth cultures. They introduced the intravenous route 
of injection, finding that the immunity to subsequent infection ap- 
peared far more rapidly than after subcutaneous injection — in 
two to three days against fourteen days. They found that duration 
of immunity varied from twenty-one days to more than six months. 
Their greater contribution was the observation that the young of 
immunized mother rabbits were usually passively protected, and 



22 BIOLOGY OF PNEUMOCOCCUS 

this observation naturally led the authors to test the curative 
value of the serum of actively immunized rabbits. The direct in- 
jection of such serums into the blood stream of the rabbit was ef- 
fective against a lethal dose of pneumococci injected twenty-four 
hours later. 

The Klemperers presented an array of new facts which, disre- 
garding certain misinterpretations, have withstood the test of time 
and supply a rational basis for the treatment of human beings with 
immune serum. They observed that the serum of pneumonia pa- 
tients after crisis conferred protection upon rabbits, that the pro- 
tective substance came into action at the beginning of crisis, and 
that it neutralized the harmful properties of Pneumococcus with- 
out destroying the antigenicity of the organism. Having tested the 
harmlessness of immune serum on themselves, they treated six 
pneumonia patients with subcutaneous injections of only four to 
six cubic centimeters, obtaining a fall of temperature within six to 
twelve hours after injection, the temperature in two cases remain- 
ing normal. The Klemperers sought to explain the action of serum 
on an antitoxic basis, but in the light of our present knowledge it 
would seem doubtful if the effect was a specific one. 

From bacteria-free bouillon cultures of virulent organisms, by 
repeated alcohol precipitation and re-solution of the precipitate in 
water, they isolated a protein substance, thought by them to be a 
pneumotoxin, which was poisonous for rabbits, withstood a tem- 
perature of 60°,* and in proper doses was capable of raising the 
resistance of the animals above the normal threshold of infection. 
In the blood of the immune animals they detected a substance not 
there previously, which it was conjectured must have been formed 
in the interval between the act of injection and the appearance of 
immunity, and which must be dependent upon the action of the in- 
jected albumen. The newly found substance in blood had no killing 
effect on living pneumococci but inhibited their toxic action. In an 

* Throughout this text the figures given for temperatures represent degrees 
on the Centigrade scale, and the initial "C" will be omitted. 



HISTORY OF PNEUMOCOCCUS: 1890-1900 23 

attempt to isolate this new curative substance, the Klemperers pro- 
gressed far enough to be able to say that it also was a protein. 
They believed that they had found an antitoxin for Pneumococcus, 
but that mistake is of no importance in view of the value of their 
major contribution. 

The work of the Klemperers gave new impetus to investigation. 
Here was an alluring and promising field ; there loomed the possi- 
bility of a biological cure for pneumonia. Emmerich and Fowit- 
zky, 857 still using the subcutaneous route for the injection of 
attenuated pneumococci, obtained only partial immunity in rabbits. 
However, when they injected diluted, fully virulent cultures intra- 
venously, the resistance appeared to be complete. Instead of using 
the serum of these animals for protective or curative experiments, 
they used the filtered juices expressed from the tissues. The fluids 
so obtained were claimed to possess idealer Heilkraft but, although 
Emmerich and Fowitzky considered their use justifiable for human 
therapy, they employed the immune tissue extracts only in animal 
tests. Bonome (1891) 137 tried sterile filtrates of bouillon cultures 
injected every other day subcutaneously, intravenously, and intra- 
peritoneally into rabbits and found that immunity appeared as 
early as two or three days after the final injection. The toxicity of 
the filtrates was directly related to the virulence of the strain, some 
animals showing local reactions, others none. He employed, with 
like effect, blood and spleen from mice killed by a culture insuffi- 
ciently virulent to kill rabbits. The defibrinated blood of the 
treated rabbits injected into the peritoneum of other rabbits pro- 
tected them against lethal doses of the toxic filtrate, but only when 
the blood was administered just prior to the injection of the fil- 
trate or culture. 

Kruse and Pansini 763 likewise produced immunity with daily in- 
jections of sterile filtrates of broth cultures. They attributed the 
protective properties of the blood of the immune rabbits to bac- 
tericidins, and although they observed phagocytosis, this was held 
to be a secondary factor. In their paper, apart from the immuno- 



24 BIOLOGY OF PNEUMOCOCCUS 

logical data, there is the account of a phenomenon, the meaning of 
which the authors failed to realize, but which much later was to 
assume wide biological significance. Studying forty-six strains of 
diplococci of pneumonic origin under varying cultural conditions, 
Kruse and Pansini noticed that the organisms when subjected to 
unfavorable media, with succeeding generations began to differ 
from the parent strain in morphology and colony formation. The 
variations ran the gamut from typical Diplococcus lanceolatus to 
Streptococcus pyogenes. Along with the changes in form there 
took place loss of capsule and of virulence, although animal pas- 
sage restored the original characters. Because of the loss of 
capsule, diminution of virulence, and the appearance of chain 
formation, Kruse and Pansini concluded that they had effected a 
mutation of Pneumococcus into Streptococcus, and that both spe- 
cies arose from a common saprophytic, streptococcal form. What- 
ever may have been their conclusions, it seems certain that they 
were observing the phenomenon of bacterial dissociation. 

A notation of Metchnikoff's 894 was undoubtedly the first record 
of the agglutination of pneumococci by immune serum. He wrote 
that the microbe of pneumonia formed very long plaquettes of 
"streptococci" in the serum of vaccinated rabbits. 

Foa and Carbone (1891), 463 continuing their work with alcohol 
and ammonium sulfate precipitates from culture filtrates, learnt 
that the immunity induced by their preparations was less enduring 
than that evoked by the untreated filtrates. Foa's later studies 
(1893) 459 were, in a way, less productive, since, unknowingly, he 
was apparently dealing with both pneumococci and meningococci. 
He did show, however, that these two cocci differed from each other 
in biological characters, and that the serum of animals immunized 
against the one species was inactive against the other. 

Here, in the matter of time and because of its bearing on the dis- 
cussion in some of the preceding pages, there may be interpolated 
the final description which Sternberg (1892) 1321 gave to Pneumo- 
coccus under the title, Micrococcus pneumoniae crouposae : 




Courtesy of Wiener Medizinischc W 'ochenschrift 



ANTON W. WEICHSELBAUM 1845-I92O 



HISTORY OF PNEUMOCOCCUS: 1890-1900 25 

Discovered by the present writer in the blood of rabbits inoculated 
subcutaneously with his own saliva in September, 1880; by Pasteur in 
the blood of rabbits inoculated with the saliva of a child which died of 
hydrophobia in one of the hospitals of Paris in December, 1880; identi- 
fied with the micrococcus in the rusty sputum of pneumonia, by com- 
parative inoculation and culture experiments, by the writer in 1885. 
Proved to be the cause of croupous pneumonia in man by the researches 
of Talamon, Salvioli, Sternberg, Fraenkel, Weichselbaum, Netter, 
Gamaleia, and others. 

Mosny (1892), 932 unconcerned about priority, turned his atten- 
tion to immunological experiments. He grew virulent pneumococci 
in broth, heated the cultures at 60°, and then filtered them. The 
subcutaneous or intravenous injection of the filtrates into rabbits 
brought about immunity four days later, but the immunity was of 
a low order. The serum of the rabbits so treated gave protection 
only when injected before or at the same time with the inoculated 
culture. Mosny could detect no bactericidal action of the immune 
serum. The cocci, on the contrary, grew fully as well in immune as 
in normal serum. In watching the growths he noted a change in the 
physical appearance of the immune serum-culture mixture which 
we now know must have been agglutination. Mosny, like Klebs, and 
more particularly Metchnikoff, saw something new and told of it, 
unconscious that he had made a discovery. 

The toxin idea was then current, and Issaeff, 673 using sterilized 
broth cultures of virulent pneumococci and chloroform and glyc- 
erol extracts of infected blood, interpreted the effects following 
their intravenous injection as being due to a toxin, but frankly 
confessed that the immune serum so obtained had no antitoxic 
properties ; neither did it show any bactericidal action. The serum 
did, however, promote phagocytosis, and Issaeff quite rightly con- 
cluded that phagocytosis played a most important part in im- 
munity to Pneumococcus. Emmerich 856 also entertained the toxin 
idea, but instead of heating or filtering broth cultures, injected 
them in diluted state. He stressed the desirability of using virulent 
strains, as well as the necessity of taking blood only from highly 



26 BIOLOGY OF PNEUMOCOCCUS 

immunized animals. Failure to do this would explain the unsatis- 
factory results of Foa, and the Klemperers. Emmerich looked upon 
the action of the immune serum as antibacterial rather than anti- 
toxic, and came near the truth when he explained the action as a 
combination of two proteins, globulin from the blood combining 
with a poisonous substance, probably also protein from the bac- 
terial cell. He, like Fraenkel and Sternberg, made a bid for preced- 
ence, objecting to Foa's claim of being the originator of serum 
therapy. 

In 1896, Washbourn, 1486 apparently unaware of Mosny's origi- 
nal observation, thus described the effect of adding pneumococci to 
specific immune serum: "When protective serum is inoculated it 
appears perfectly clear at the end of twenty-four hours, but at the 
bottom a sediment is seen. The sediment consists of pneumococci 
staining well and grouped in masses." 

Metchnikoff's, Mosny's, and Washbourn's unnamed phenomenon 
was verified in 1897 by Bezancon and Griffon 108 ' 9 and called by 
them "agglutination." They found that the serum of patients dur- 
ing pneumococcal infections acquires agglutinative power, and 
from their experiences they drew the conclusion, "that from the 
standpoint of agglutination there exist several races of pneumo- 
cocci, which behave as though different microbes." Here was the 
basis for a method of serological classification, neglected for thir- 
teen years until Neufeld and Haendel made it their own. 

Eyre, with Washbourn (1896), 373 like Kruse and Pansini, was 
also close to the phenomenon of bacterial dissociation. 

In old broth cultivations the majority of cocci are dead, but a few re- 
sistant forms remain living; and, by transplanting a sufficient quantity 
into fresh media, growth occurs and a new generation arises. We have, 
moreover, found that this second generation differs in morphology, 
biology and pathogenic properties from the parent stock. It in fact rep- 
resents a distinct variety, possessing practically no virulence, and 
growing luxuriantly, even at 20° C, on all the usual media. 

Their first attempts to bring about reversion failed, mainly 



HISTORY OF PNEUMOCOCCUS: 1890-1900 27 

because they passed the strain through eggs. Finally, however, pas- 
sage through a rabbit restored the degenerated forms to the origi- 
nal state of the parent strain. Without venturing an explanation, 
Eyre and Washbourn concluded, "Our experiments are in favor of 
the theory . . . that the individual cocci or their descendants ac- 
tually alter in character under varying conditions," or, to use the 
modern term, dissociate. 

The quest for a potent immune serum was continued by Denys 
(1897), 312 who met with greater success than did his predecessors. 
Denys first raised the virulence of pneumococcal cultures by serial 
passage of infected blood through rabbits, then administered both 
heated and filtered cultures to normal animals, at first rabbits, 
then goats, and later horses. After this preliminary treatment, 
Denys gave a series of injections of unheated cultures, then of the 
blood of infected rabbits, and finally of living, virulent pneumo- 
cocci. The serum thus obtained prevented infection, was curative, 
and neutralized in rabbits the alleged "toxins" of Pneumococcus. 
The results of controlled experiments convinced Denys that the 
immune serum so prepared was not bactericidal, but that it 
contained a substance which stimulated the white corpuscles to 
phagocytosis, or as he phrased it, Uimmunite du lapin contre le 
pneumocoque, a sa source dans une modification de son serum; and, 
L'element immunisant primordial est le serum et le leucocyte par 
lui-meme n*est rten. 

Pane, 1044 in the same year, not only obtained potent serums from 
rabbits, cows, and asses, but tested their curative action on human 
beings. Of twenty-three pneumonia patients treated by intravenous 
injection, only two died. Pane, incidentally, noted definite aggluti- 
nation in the test tube and phagocytosis in the blood and therefore 
was inclined to accept MetchnikofPs theory as affording an expla- 
nation for the therapeutic action of the serum. 

Mennes 893 fully agreed with Denys' idea that it was the immune 
serum and not the normal or immune leucocyte which stimulated 
phagocytosis. He, also, immunized horses, gave the serum intra- 



28 BIOLOGY OF PNEUMOCOCCUS 

venously to pneumonia patients, and favored large doses. In the 
same year, a few months earlier as a matter of fact, Washbourn 1487 
had injected ponies with a culture of exalted and constant viru- 
lence, and after proving to his satisfaction the favorable action of 
the serum on rabbits, administered it to six pneumonia patients. 
All, although severely ill with the disease, recovered. He made an- 
other advance by standardizing the serum, and set as a unit the 
smallest amount of serum which, when mixed with ten times the 
least fatal dose of pneumococci and injected into the peritoneum, 
would bring about the survival of the test rabbit. Washbourn em- 
phasized the necessity of early treatment and advised injections of 
at least six hundred "units" twice daily. By himself, and in the 
following year with Eyre, 876 he examined immune horse serum for 
protective properties, and concluded that there was no parallelism 
between agglutinative, bactericidal, and protective power. In their 
papers ( 1899-1900), 375,377 these two authors gave further reports 
on the results of potency tests made on antipneumococcic serum, 
including two samples of Pane's product. By this time they had 
modified their method, and now injected the serum intravenously 
and then the culture intraperitoneally into rabbits. The serums, in 
one cubic centimeter amounts, usually protected rabbits against a 
thousand or more fatal doses of pneumococci, but in a few in- 
stances failed to protect against strains from another source, ow- 
ing, as we now know, to a lack of type correspondence. 

Summary 
At the turn of the century the results of Pneumococcus investi- 
gations could be inventoried as follows: Pneumococcus was ac- 
cepted as the causative agent in lobar pneumonia ; it could be 
grown outside the body, and some of its habits and metabolic ac- 
tivities were becoming known ; Pneumococcus or its products were 
found to raise the resistance of some experimental animals to ho- 
mologous infection ; these animals could be made to yield a serum 
capable of conferring passive protection on vulnerable or stricken 



HISTORY OF PNEUMOCOCCUS 29 

creatures ; and such a serum gave an early promise of mitigating 
pneumococcal affections in man. 

The first glimpses of the vagaries of microbic behavior under 
varying environment were later to expand into broader views of 
that instability of bacterial characters which result in dissociation 
into varied and distinct forms or possible mutation into alien spe- 
cies. These discoveries were to clarify our ideas concerning viru- 
lence and the consequent infectiousness of a bacterium for a sus- 
ceptible host. The fact that the serum from animals treated with 
Pneumococcus or some of the derivatives of Pneumococcus pos- 
sesses the property of clumping this organism was, at the hands of 
Neufeld, Dochez, and Gillespie, and later of Cooper and her asso- 
ciates, to form the basis for a method of separating the members 
of a supposedly homogeneous species into thirty or more clearly 
defined and serologically specific types. This biological classifica- 
tion was to give a new aspect to the problems perplexing the epi- 
demiologist, the bacteriologist and immunologist, and the clinician. 

For a time Pneumococcus investigation was to lag, and then, 
stirred by the contributions of Neufeld and of the Cole school, it 
was to receive a new impetus, and to bring the vital activities of 
this amazing cell more clearly into sight. The facts that have been 
disclosed in this new and closer view, their bearing on general bio- 
logical problems, and their application to the development of pre- 
ventive and curative agents will be related in subsequent chapters. 



CHAPTER II 
BIOLOGY OF PNEUMOCOCCUS 

The morphology of Pneumococcus in tissues and in cultures; iso- 
lation, cultivation, and preservation; viability and fragility, and 
sensitiveness to bile, soaps, and other chemical substances. 

For the student of Pneumococcus there is no lack of reliable de- 
scriptions of its general or detailed biological features. In or- 
der, however, that the student may not be obliged to go beyond the 
covers of this volume for information about the organism, its inti- 
mate features will be presented here. The more important points 
have been freely taken from the clearest and most accurate ac- 
counts in current text and reference books, and acknowledgement 
is made to the respective authors.* 

Morphology 

The anatomy of Pneumococcus is simple and distinctive. It is 
best studied in preparations made from body fluids of man or ani- 
mals suffering from infection. Next best are young, vigorous cul- 
tures grown on proper culture media containing blood, serum, or 
other body fluids. When typical, the organism consists of a pair of 
oval or lance-shaped cocci, their somewhat flattened proximal ends 
in apposition and their distal portion pointed. Sometimes single 
cocci are seen, while at other times single or paired cocci may be 
arranged in short or even long chains, resembling a string of beads 
— the chapelet originally described by Pasteur. There may be 
many variations from this characteristic form even when the envi- 
ronment is favorable. The individual cocci may be round and of 

* The sources consulted, besides original papers, are the article by Neufeld 
and Schnitzer in the third edition of the Kolle-Kraus-Uhlenhuth Handbuch der 
Pathogenen Mikroorganismen;?* Zinsser and Bayne-Jones, Textbook of Bac- 
teriology ,1579 Acute Lobar Pneumonia (Rockefeller Institute Monograph, 
No. 736) ; and A British System of Bacteriology in Relation to Medicine^ 



BIOLOGY OF PNEUMOCOCCUS 31 

varying size (0.5 to 1.25|j), or they may be so elongated as to re- 
semble bacilli. One authority describes the cells as "small," another 
as "rather large," but the actual size varies with the circumstances 
under which the organism is observed. In any given preparation, 
along with typical forms, other members displaying every degree 
of involution or degeneration may be present, while in aged cul- 
tures aberrant forms may be the rule. 

Pneumococcus has no spores, no vacuoles, no visible granules, no 
flagella, and is non-motile. It reproduces by the primitive method 
of transverse fission. 

The most distinctive morphological feature of the organism is 
the capsule. The capsule is more prominent when the cocci are ex- 
amined in the body fluids of infected animals, or in media enriched 
with body fluids, becoming faint or disappearing when the strain is 
grown on less rich substrates. The capsule envelops the single, 
paired, or chained cocci, frequently with a uniform periphery, al- 
though sometimes it shows indentations between the twin cells or 
between the paired individuals in chains. 

STAINING METHODS 

Pneumococcus is readily stained with the usual aniline dyes and 
is Gram-positive. Exposure to the action of specific immune serum, 
or even to acidulated pepsin or alkalinized trypsin mixtures fails 
to rob the cocci of this property (Wilke 1520 ). Only when they are 
subjected to the digestive action of leucocytes, or after death and 
partial disintegration in old cultures, do they give up the dye to 
decolorizing agents. For peritoneal fluids, sputum, and cultures 
the Gram stain is generally used. The authors prefer Burke's 
modification 191 to that of Sterling* or to the original formula. 545 

Dold 324 " 5 recently devised a staining method by which he claimed 
to be able to divide Gram-positive cocci into four groups and to 
demonstrate tinctorial differences between pneumococci and strep- 
tococci. He treated preparations made from cultures on solid me- 

* Original reference unknown. 



32 BIOLOGY OF PNEUMOCOCCUS 

dia, stained by an aniline dye, phenol, and iodine, with a mixture 
of one part 40 per cent aqueous solution of urea and nine parts 
absolute alcohol. Organisms which retained the dye were called 
positive; those from which the dye was extracted were negative; a 
third class exhibited fluctuations in their ability to retain the ani- 
line color; while the fourth group displayed periodic changes from 
positive to negative and vice versa. He reported that all strains of 
streptococci tested were without exception negative, while Strepto- 
coccus mucosus and thirty-nine strains of pneumococci showed a 
change from positive to negative or vice versa. Some of the latter 
gave predominantly positive results while some were usually nega- 
tive with only an occasional positive reaction. These latter he con- 
sidered as transition forms between the predominantly positive 
pneumococci and the uniformly negative strain of Streptococcus. 
For demonstrating pneumococci in tissues the method described 
by Wadsworth* may be used to advantage, while the Gram stain 
is to be preferred to the Gram-Weigert technique.f 1506 

THE CAPSULE 

Modern chemical study of the capsular material has given a ra- 
tional basis for the serological classification of all pneumococci 
into definite and specific types. It is this peculiar and complex com- 
ponent of the pneumococcal cell that determines its specific anti- 
genic stimulus and its immunological behavior in the presence of 
antibodies. This knowledge enables us to utilize in a far more in- 
telligent way Pneumococcus or its components in the induction of 
active immunity and consequently in the production of immune 
serums, and furnishes us with a delicate reagent for measuring the 
body's response to various immunizing procedures, and for deter- 
mining the potency of antipneumococcic serums. 

The capsule interested and perplexed many of the earlier investi- 
gators. Described first by Pasteur 1066 as an aureole, and by Fried- 

* Zinsser and Bayne-Jones.1579 

f Neufeld prefers the Gram technique; Zinsser the Gram-Weigert technique. 



BIOLOGY OF PNEUMOCOCCUS 83 

lander 486 " 7 as a Kapsel, it was later called a rim, or a hull, and 
its presence undoubtedly contributed to the confusion between the 
cocci first described by Friedlander and the well-known bacillus 
that bears his name. Because of the inconstancy of its appearance 
particularly under different conditions of artificial cultivation, the 
belief arose that it was not a diagnostic feature of the species. 
Pane 1045 went so far as to assert that it was a degenerative and not 
a developmental phenomenon. Friedlander had always contended, 
however, that the capsule was the product of a vital function of 
the cell. 

Aoki 18 noticed that capsule formation proceeded more rapidly 
at body than at lower temperatures, and that the capsules were 
larger and more distinct when the cocci were grown in immune se- 
rum or in media containing such serum. This was the Quellung ef- 
fect utilized by Neufeld and Etinger-Tulczynska 988 in their re- 
cently published method for type determination. The capsule was 
often described as gelatinous but, since the time of Pasteur and 
Friedlander, it had been more generally believed to consist of mu- 
cin. It was Preisz 1108 who, acting upon this assumption, was the 
first to demonstrate capsular material in the blood and other body 
fluids of animals succumbing to pneumococcal infection. He be- 
lieved that a general, fatal invasion of the body by these cocci 
could occur only when the accumulation of mucin — now soluble 
specific substance — in the body had reached a certain level. 

STAINING THE CAPSULE 

Many methods have been devised for demonstrating the capsule 
of Pneumococcus. The first record of a capsule stain for Pneumo- 
coccus is that by Friedlander (1885) 491 who, after fixing the prepa- 
ration in the flame, dipped it for a few minutes in one per cent ace- 
tic acid. After blowing off the acid he dried and then stained the 
film by a few seconds' exposure to a saturated aniline-water gen- 
tian violet solution. MacConkey (1898) 843 recommended staining 
with dahlia, methyl green, and a saturated alcoholic solution of 



34 BIOLOGY OF PNEUMOCOCCUS 

fuchsin. The method, he claimed, gave a clear image suitable for 
photography. Hiss 650 in 1902 gave two procedures, the first, a gen- 
tian violet-potassium carbonate method, the other, and a better 
one, a preliminary treatment with gentian violet or fuchsin fol- 
lowed by washing with 20 per cent copper sulfate solution. The 
Welch* formula of glacial acetic acid, followed by aniline-water 
gentian violet gives clear pictures of the capsule. These methods 
are widely used and give satisfactory results. 

Buerger (1905) 168 described a different and also reliable proce- 
dure, but the intricacy of the technique prevented general adoption. 
Malone, 860 after a preliminary staining with Congo red, treated 
the film with dilute hydrochloric acid in alcohol, counterstaining 
with methyl violet. Malone claimed that by this technique he 
was able to demonstrate capsules when the Hiss method failed. 
Wherry 1515 mixed a suspension of pneumococci in diluted serum 
with an equal amount of 0.05 normal hydrochloric acid, spread the 
mixture in a thin film, fixed the film with heat, and stained it 
quickly with carbol gentian violet. Leifson 797 applied to films a 
mixture of a saturated aqueous solution of ammonia or potassium 
alum, tannic acid in water, 95 per cent ethyl alcohol, and saturated 
alcoholic solution of basic fuchsin. He followed this treatment with 
methylene blue in aqueous borax solution. This method never came 
into general use. The Huntoon* technique is suitable only for 
cultures and not for animal exudates. India ink is the choice of 
some bacteriologists. It is a negative stain, in that the medium on 
the slide is colored black, while the cells and the capsule are un- 
stained. Levy-Bruhl and Borin, 805 however, reported that this stain 
outlined the capsules on Type III organisms when no albuminous 
substances were present in their "T" medium, but failed with other 
types of pneumococci grown on this special substrate. For general 
purposes, however, the Burke or Sterling modifications of the 
Gram stain suffice, while the Gram stain, the Gram-Weigert, or the 

* Zinsser and Bayne-Jones.is™ 



BIOLOGY OF PNEUMOCOCCUS 35 

method described by Wadsworth* may be used to demonstrate 
capsules in tissue sections. 

The capsules are indicative of the vigor and virulence of the 
strain. They may best be seen "in the blood, serum and inflamma- 
tory exudate of the infected rabbit and white mouse. They are 
equally well marked in the fresh sputum of pneumonia patients, 
especially in the early stages of the disease, and in the exudate ac- 
companying such pneumococcus infections as meningitis, otitis 
media and empyema."* 

The addition of blood, serum, milk, or other body fluids to nu- 
trient media favors the development of the capsule under artificial 
cultivation. 

Isolation of Pneumococcus 

ANIMAL INOCULATION 

One of the simplest ways of isolating pneumococci from infected 
material is by mouse inoculation. If the source material is sputum, 
a small portion can be washed in sterile broth or saline solution in 
a Petri dish to remove the majority of accompanying mouth or- 
ganisms. The washed sputum together with a small quantity of 
broth is emulsified by grinding in a sterile mortar and the suspen- 
sion in amounts of 0.5 to one cubic centimeter injected into the 
peritoneal cavity of a white mouse. Just before or shortly after 
death of the animal, the heart is exposed and, under sterile precau- 
tions, punctured with a capillary pipette or hypodermic needle, 
and the blood aspirated and planted in broth and on blood-agar 
plates. In this way a pure culture can usually be obtained at the 
first trial. Pieces of lung or other infected material may be treated 
in the same manner as sputum. When it is suspected that few pneu- 
mococci are present, the tissue may be macerated in broth and 
given a preliminary incubation before the mouse injection. f 

* Zinsser and Bayne-Jones.is™ 

t For complete details of the method see Appendix. 



36 BIOLOGY OF PNEUMOCOCCUS 

DIRECT CULTURES 

Instead of the practice of animal inoculation, washed sputum, 
blood, or broth suspensions of infected material may be streaked 
on plates poured with fresh blood agar or serum-dextrose agar. 
For the cultivation of organisms directly from the circulating 
blood, the blood withdrawn by venous puncture may be added to a 
fairly large volume of dextrose broth in flasks, or mixed with mol- 
ten dextrose agar and poured into Petri dishes. Pneumococcus, be- 
ing facultative in its oxygen needs, grows on the surface as well as 
in the depths of the medium. 

From agar plates single colonies of pneumococci may be picked 
and replanted on agar, seeded on serum or blood-agar slants or in 
serum or glucose broth. These media serve for the further propa- 
gation of the original broth cultures when pure. Small inoculums 
grow more successfully on solid than on liquid media. Incubation 
should be carried on at a temperature of about 37.5° ; 25° and 41° 
represent the lower and upper limits favorable to growth. A rarity 
were the strains isolated from blood and exudates by Eaton* in 
Zinsser's laboratory, which grew only at 25°. At 37.5° the organ- 
isms failed to grow unless incubated in an atmosphere containing 
about 10 per cent of carbon dioxide. A somewhat similar strain 
was that previously reported by Kindborg. 712 Isolated from rusty 
sputum by mouse inoculation, the organism displayed all the char- 
acters of a typical pneumococcus, yet it grew readily at 22° and 
rapidly and energetically liquefied gelatin. While pathogenic for 
mice, it was avirulent for rabbits. 

Cultivation 

Pneumococcus, being a strict parasite, is somewhat fastidious in 
nutritional requirements, as well as sensitive to the physical and 
chemical conditions of its surroundings. The essential ingredients 
of culture media are meat extractives prepared from fresh muscle 

* Quoted by Zinsser and Bayne-Jones.i579 



BIOLOGY OF PNEUMOCOCCUS 37 

tissue, protein of comparatively small molecular size in the form of 
peptone, a small amount of sugar, mineral salts, and a suitable 
concentration of hydrogen ions. All media should be subjected to 
the least degree of heat necessary to effect extraction of the meat, 
to render the ingredients soluble, and to ensure final sterility. 

The earlier bacteriologists employed the only media known in 
their time, such as egg albumen (Klebs 718 ), veal or beef broth 
(Pasteur), then broth with the addition of gelatin or agar (Fried- 
lander 487 ) and later of blood (Nissen, 1013 and Gilbert and Four- 
nier, 513 among others), serum, or other body fluids. Fraenkel 469 
employed milk, but finding the cocci lost virulence on this medium, 
turned to coagulated blood serum and then to meat-infusion agar. 
Eggs, both in the shell (Bunzl-Federn 186 ), inspissated, and as a 
base with broth (Gaskell 499 ) have been employed for the mainte- 
nance of virulence, while Grawitz and Steffen 547 preferred co- 
agulated sputum as a favorable medium for capsule formation. 
Schmidt 1236 also recommended sputum as a medium on which 
Pneumococcus developed clear, well-formed capsules, and which 
also would restore capsules to cocci that had lost them after culti- 
vation on agar. Gilbert and Fournier 513 found defibrinated blood of 
man and other animals to be suitable for the propagation of pneu- 
mococci. Carnot and Fournier 200 noted constancy of virulence and 
capsule formation of strains grown in broth and on agar contain- 
ing brain tissue, but the fluid medium could be employed only for 
the carrying on of pure cultures. 

Among the great variety of nutrient materials recommended, 
time and experience have shown the essential ones, and these are 
now incorporated into a few formulas which meet all the needs of 
the student of Pneumococcus.* While Pneumococcus can be grown 
within a fairly wide range of cultural conditions, the most vigor- 
ous and viable individuals are obtained when careful attention is 
given to the needs peculiar to this organism. Fresh beef, veal, or 

* See Appendix. 



38 BIOLOGY OF PNEUMOCOCCUS 

horse muscle, freed from fat, furnish a better base than the com- 
mercial meat extracts. Peptone preparations containing the higher 
proportion of proteose nitrogen, mainly because of the stronger 
buffering action, are to be preferred to preparations in which the 
bulk of the nitrogen is in the form of peptones and amino acids. 
The latter, however, because of their lower cost may be substi- 
tuted for routine purposes. In different formulas the amount of 
peptone varies from 0.1 to 2 per cent, depending upon the pro- 
teose nitrogen content of the preparation. Felton and Dougherty 425 
claimed that 2 per cent of peptone preserves and increases viru- 
lence. 

Kruse and Pansini 763 first drew attention to the fact that certain 
preparations of peptone hindered full growth, a problem later 
studied by Wright 1549 in 1933, who pointed out the fact that one 
of the difficulties in preparing suitable broth was largely due to in- 
complete reduction of peptone. This obstacle could be overcome by 
adding the peptone to the broth before heat is applied, thus ex- 
posing it to the reducing action of meat or meat infusion during 
the steaming process. The inhibitory action of peptone may be fur- 
ther diminished by the reducing action of sugars and an alkaline 
reaction (Wright). Dubos 3334 also found that some commercial 
peptones contain bacteriostatic substances. These may be removed 
by precipitation with acid and acetone, or their inhibitory action 
may be neutralized by the addition of reduced thiol compounds 
(0.0003 per cent thiogly collie acid). Reduced cysteine increases 
the rate of growth. 

A small amount of sodium chloride, usually 0.5 per cent, ap- 
pears to be a necessary ingredient, although sodium phosphate 
may be substituted. An excess of salt retards or prevents the 
growth of Pneumococcus (Wright 1548 ), and its concentration, 
whether as chloride or phosphate, should not exceed 0.1 molar 
(Dernby and Avery 313 ). Glucose stimulates and enhances initial 
growth, and quantities ranging from 0.1 to 4.0 per cent have been 
advised, while Turro 1430 preferred 8 per cent ! Amounts higher than 



BIOLOGY OF PNEUMOCOCCUS 39 

one per cent, because of the glycolytic action of Pneumococcus 
with the resulting acid production, may produce retardation 
or complete inhibition of growth and bring about autolysis 
(Wright 1548 ). The presence of muscle sugar in the meat base must 
be reckoned as an additional sugar factor when fermentation reac- 
tions are to be quantitatively measured. McGuire, Valentine, Whit- 
ney, and Falk 879 advised titration of the fermentable reducing 
sugar in muscle (beef heart) infusion and, taking the equivalent of 
0.5 milligram of glucose per cubic centimeter as the lower limit, 
then added glucose to the desired concentration. Glycerol may re- 
place glucose or other sugars as a source of carbohydrate mate- 
rial, but glycerol, like the other sugars, is susceptible to the 
fermentative action of Pneumococcus and yields acid on enzymatic 
cleavage. 

The acid formed in broth by the action* of Pneumococcus may 
be neutralized either by the addition of sterile, powdered calcium 
carbonate (Wurz and Mosny, 1554 and Hiss 650 ), or of small pieces 
of marble, washed and placed in the test tubes before filling and 
sterilization (Bolduan 135 ). 

The initial hydrogen ion concentration of the medium and the 
changes occurring during growth are vital factors in the proper 
cultivation of the organism. According to Dernby and Avery, 313 
culture media should have an initial reaction represented by a pH 
of 7.8 to 8.0 with an optimal pH of 7.8. They gave the limiting hy- 
drogen ion concentration for initiation of growth for the various 
types of Pneumococcus as pH 7.0 to 8.3. Strains will live but will 
not actively multiply in substrates having a degree of acidity or 
alkalinity greater than those represented by these figures. Lord 
and Nye 829 have given further information regarding the relation 
of hydrogen ion concentration to the life, growth, and death of 
Pneumococcus. As the medium becomes more acid (pH 7.4 to 6.8) 
the organism may live many days; between 6.8 and 5.1 death be- 
gins to take place — the greater the acidity, the more rapid the 
death, while at 5.1 the cocci die within a few hours. In cloudy sus- 



40 BIOLOGY OF PNEUMOCOCCUS 

pensions of washed pneumococci dissolution is most marked within 
the range pH 5.0 to 6.0. Some disintegration occurs toward the 
alkaline side but none at the most acidic end of the scale. 

For the successful initiation of growth in broth, once a pure cul- 
ture is obtained, a fairly heavy amount of inoculum is advisable. 
Cole used 0.1 cubic centimeter for every cubic centimeter of broth. 
These amounts are obviously impossible when cultures are to be 
planted from single colonies or from mouse-hearts' blood and simi- 
lar materials. When original infectious material is used as inocu- 
lum a small amount usually suffices. A generous planting, however, 
favors multiplication. 

The cycle of pneumococcal growth in broth has been studied by 
Chesney. 220 When broth is inoculated with a culture that has passed 
the stage of maximal growth, there ensues a latent period before 
growth appears in the freshly seeded broth. When, however, inocu- 
lation is performed with cultures at their maximal rate of growth, 
there is no delay in multiplication. This initial latent period or 
lag — that is, the interval between the time of seeding and the time 
at which maximal rate of growth begins — is followed by a phase of 
rapid growth, during which the organisms are dividing regularly. 
This stage is followed by a stationary period, in which the organ- 
isms cease to multiply at maximal rate so that the increase in 
number becomes slower and finally ceases and, although they re- 
main viable, the number of cells present in a unit volume remains 
approximately constant for an appreciable length of time. Finally 
the period of decline sets in, in which the number of living organ- 
isms begins to decrease. Chesney believed that this lag is an expres- 
sion of injury which the bacterial cell sustains from previous envi- 
ronment. 

The failure of pneumococci to continue to grow and multiply 
after the stationary period is due to the production of acid or pos- 
sibly of other inhibiting substances. Morgan and Avery 915 listed 
the inhibiting factors as the accumulation of acid products, the 
exhaustion of nutritive substances, and the formation and accumu- 



BIOLOGY OF PNEUMOCOCCUS 41 

lation, under certain cultural conditions, of peroxide in the me- 
dium. Hager,* by daily neutralizing the newly formed acid has 
succeeded in prolonging the period of growth and, therefore, in ob- 
taining unusually heavy yields of pneumococci from a single lot of 
broth. 

Felton and Dougherty 423 " 4 contrived an ingenious apparatus for 
delivering a continuous supply of pneumococci at the stage of most 
active growth. By an automatic device, an inoculum of vigorous, 
young culture was removed from the exhausted medium and 
planted in a flask containing a fresh food supply of milk. The 
transfers were made at two, four, and eight-hour intervals. Propa- 
gation by this method at eight-hour intervals raised the virulence 
of the strain to a degree unprecedented in any former in vitro ex- 
periments. Methods for obtaining large amounts of living pneu- 
mococci for chemical study as well as for other purposes have 
been described by Felton and Huntoon, 667 while that of Hager has 
not yet come to publication. f 

These media, varying in their constituents, all require heavy 
inoculation with a vigorous culture (Gillespie 515 ), careful initial 
adjustment or subsequent correction of the reaction and, of 
course, incubation at a constant temperature of about 37.5° if 
they are to give a large yield. Dubos 331 explained the necessity for 
large amounts of inoculum in seeding broth as being based on the 
establishment of a proper reduction potential in the medium. This 
condition is brought about by the large inoculum owing to the re- 
ducing properties of the bacterial cells. 

ACCESSORY SUBSTANCES 

Optimal growth conditions, that is, as far as rate of growth, 
number, and viability of the organisms are concerned, can be pro- 
vided beyond those already mentioned by the addition of a variety 
of substances. Autoclaved gelatin, by its buffering action in addi- 
tion to its nutritive elements, has been found by Piatt 1096 to induce 

* Personal communication. t See Appendix. 



42 BIOLOGY OF PNEUMOCOCCUS 

and sustain growth of Pneumococcus in otherwise unsuitable me- 
dia. Robertson, Sia, and Wood 1149 ascertained that the protective 
action of gelatin lies largely in shielding pneumococci against 
physical injury — possibly the toxic action of electrolytes — which 
occurs during dilution in solutions of crystalloids. Furthermore, 
gelatin exerts a well-marked preservative action of unknown na- 
ture in protecting the organisms against early dissolution. 

One of the most commonly used substances is blood, of either 
man, rabbit, horse, or sheep. Pasteur, Sternberg, and Levy and 
Steinmetz 802 early recognized its value. Citrated, laked, or, better, 
defibrinated blood augments the growth-promoting value of the 
substrate. It is best used in a ratio of one part or less to ten to 
twenty parts of medium. Normal serum, transudates, and ascitic 
and hydrocele fluids similarly supply elements favorable to the 
growth of Pneumococcus, and are common ingredients in many 
media formulas (Fraenkel, 468 Behring and Nissen, 98 Mosny, 933 Le- 
vinthal, 800 and many others). 

In a study of the growth-promoting properties of serum from a 
variety of animal species, Bezancon and Griffon 112 found that, in 
general, growth was more rapid and abundant in the serum of ani- 
mals susceptible to pneumococcal infection. However, in these se- 
rums cell death took place rapidly after maximal growth had been 
reached. In the serum of resistant animals, on the other hand, 
growth was meager, although the organisms remained viable and 
virulent for a longer period of time. The serum of young animals, 
whether of a sensitive or refractory species, possessed the growth- 
promoting properties of susceptible species, whereas the serum of 
older animals was less favorable to growth. 

Bordet 139 has recently described an unexplained and as yet un- 
confirmed cultural and morphological appearance of cultures in 
rabbit serum. The growth of pneumococci had a milky appear- 
ance, and at the bottom of the tube a sediment formed which con- 
sisted of a clustered mass of large round elements. These bodies 
failed to take well the basic stains but were readily stained by neu- 



BIOLOGY OF PNEUMOCOCCUS 43 

tral dyes. They were variable in size and had fairly regular out- 
lines and a granular structure. Inasmuch as these bodies did not 
appear in serum media previously heated at 56° for one-half hour, 
but did appear when the temperature was no higher than 50°, 
Bordet thought that their production was due to alexin, although 
they were not present in broth containing serum from rabbits vac- 
cinated against Pneumococcus, either of homologous or heterolo- 
gous type. 

Blood and serum, in addition to contributing some nutritive 
substances, act as buffers in controlling the reaction and, being 
colloids, arrest any toxic action of inorganic salts. Blood, because 
of its oxidation-reduction system, tends to maintain a proper oxy- 
gen balance, while the iron in the hemoglobin seems to act as a 
catalase. In studying the supposed necessity for hemoglobin for 
the growth of Bacillus influenzae, Thjotta 1301 discovered that this 
substance could be omitted if extracts of mucoid bacilli or of Ba- 
cillus proteus were substituted. Thjotta looked upon the accessory 
substance in such extracts as a vitamin which, in many ways, cor- 
responded to Wildier's "Bios" of yeast, the "auxomones" of Bot- 
tomly, or the hormones. Then Thjotta and Avery 1392 " 3 learnt that 
these substances — the "V" factor — alone would not support the 
growth of hemophilic bacteria through several generations, but 
that another substance, the so-called "X" factor, contained in red 
corpuscles was essential. This X factor is heat-stable and acts in 
minute amounts. Both the V and X factors are required for the 
continued and complete development of Pneumococcus as well as 
for the growth of hemophilic organisms. 

Baudisch and Dubos 89 investigated the influence of iron com- 
pounds on the viability of pneumococci. Small amounts of iron 
oxide prolong the life of avirulent strains but are less favorable to 
virulent strains. Iron may be stimulating or harmful depending 
upon the particular chemical radical with which it is combined. 

Another accessory substance of animal origin, recommended by 
Quiroga, 1115 is liver. The addition of an extract of beef liver to 



44 BIOLOGY OF PNEUMOCOCCUS 

peptone broth or Martin bouillon gives early growth with less pro- 
duction of acid than that which occurs when glucose is used. In 
such a medium it is probably the glycogen or other sugars which 
supply the stimulus, while the tissue itself and the hemoglobin fur- 
nish the other growth-promoting factors. The protective action of 
muscle tissue is exemplified in the hormone-blood agar and hor- 
mone-gelatin broth of Bailey. 67 The formula is a modification of 
that of Huntoon 684 for the preparation of original hormone me- 
dium, and both methods, and also that of Douglas for making 
tryptic digest medium, yield substrates well suited not only for the 
prolific growth of pneumococci but for conservation of essential 
biological characters of the organism. 

Still another factor to be considered is the oxygen requirement 
of Pneumococcus. The organism is a facultative anaerobe, and 
grows both in the presence and absence of oxygen. Examples of 
anaerobic strains of pneumococci have been encountered by Avery 
and his associates,* while two similar strains which apparently 
constitute two new serological types have recently been described 
by Smith. 1296 Because of its action in elaborating toxic peroxides 
in the surrounding medium, as first demonstrated by McLeod and 
Glovenlock 885 and later proved by McLeod and Gordon 881 " 4 to be 
hydrogen peroxide, Pneumococcus may hinder or entirely prevent 
its own growth. The inhibiting effect of the peroxide may be over- 
come by the addition of plant tissues or other substances exerting 
an oxidizing- reducing action (Avery and Morgan 62 ). 

VEGETABLE ACCESSORY SUBSTANCES 

Avery and Morgan 61 ' 63 found that the addition to broth of ster- 
ile, unheated plant tissue, such as yellow and white turnip, carrot, 
beet, parsnip, white and sweet potato, and banana,f not only 
caused acceleration of pneumococcal growth, but served to induce 
abundant multiplication even when the seeding was too minute to 

* Personal communication. 

f Falk, Valentine, McGuire, and Whitneysss have more recently (1932) rec- 
ommended the use of the green banana as enriching material in the place of 



BIOLOGY OF PNEUMOCOCCUS 45 

initiate growth in the medium. With potato broth the beginning 
period of growth was prolonged and cell death delayed. Moreover, 
in plant-tissue medium, the zone of hydrogen ion concentration 
within which growth could be initiated was considerably extended 
beyond the optimal range in ordinary bouillon. Fresh plant tissue, 
that is tissue which has not been subjected to heat or oxidation, 
was necessary for this effect, and this fact explains the action of 
the tissues in promoting the growth of certain anaerobic bacteria 
in the presence of oxygen. In the case of Pneumococcus, the action 
of the oxidizing-reducing system of the V and the X factors* leads 
to the destruction of toxic peroxides. 

Yeast, in the form of autolysate, in combination with clotted 
horse blood, peptone, agar, glucose, and maltose was found by 
Hitchens 654 to be a favorable medium for promoting the growth of 
pneumococci. 

APPEARANCE OF GROWTH 

In a suitable broth, the first evidence of growth is a faint, uni- 
form clouding of the medium. There is a slow deposition of a light, 
flocculent sediment at the bottom of the container. On further 
incubation there is a gradual clearing of the medium with the ac- 
cumulation of a heavier deposit, while some of the falling particles 
may adhere to the side of the vessel. There is no pellicle formation. 
Growth is more rapid and luxuriant when glucose, the accessory 
substances already mentioned, or some sterile body fluid are added. 

In milk, Pneumococcus thrives and develops capsules, and if 
transfers are made at six or eight-hour intervals, the vigor and 
virulence of the organism are maintained. Prolonged cultivation in 
this medium without frequent transplants, however, is detrimental 
to the cocci owing to the acid produced. Milk is coagulated by 

fresh blood in beef-heart broth for the cultivation of pneumococci, while Larson 
and Thompson"^ employ broth containing sterile, fresh, unheated potato ex- 
tract for growing pneumococci for type determination and bile-solubility tests. 
The latter medium has the advantage over serum or blood broth of being free 
from extraneous substances which might interfere in these tests. 
* See p. 43. 



46 BIOLOGY OF PNEUMOCOCCUS 

practically all strains. Pneumococci will grow in gelatin at 37° 
but, because gelatin melts, it is not a useful medium for propagat- 
ing pneumococci. 

On agar, Pneumococcus grows in very small, round, water-clear, 
non-confluent colonies, resembling in many respects those of strep- 
tococci, but differing from them in being more transparent, more 
moist, and flatter. Under the microscope, colonies of Pneumococ- 
cus are seen to be finely granular, with dark centers fading to 
lighter zones nearer the periphery, which is regular, sometimes 
slightly wavy, but never showing the intertwined convolutions such 
as those seen in colonies of Streptococcus. In agar stab cultures 
and sub-surface agar plate cultures, the colonies appear within 
twenty-four to thirty-six hours, are smaller, sometimes almost in- 
visible, and indistinguishable from colonies of Streptococcus. Un- 
der low magnification they show partly as yellowish-brown, partly 
as bright, oval, lenticular or whetstone-like forms. 

On blood-agar plates the colony may be surrounded by a zone of 
methemoglobin formation with an exterior zone of greenish color 
after twenty-four or more hours of incubation. Later a zone of 
hemolysis may appear at the margin fainter than that encircling 
streptococcal colonies, and this appearance may lead to error in 
the differentiation of the two organisms. When grown on "mix- 
tures of whole rabbits' blood and agar, the pneumococcus grows 
well, and forms, after four or five days, thick, black surface colo- 
nies, not unlike sun blisters on red paint. These colonies are easily 
distinguished from those of streptococci, and are of considerable 
differential value (Hiss)."* 

"Upon potato, thin, moist growth appears, scarcely visible and 
indistinguishable from an increased moisture on the surface of the 
medium," making this an unsuitable substrate. "Upon Loeffler's 
coagulated blood serum, the pneumococcus develops into moist, 
watery, discrete colonies which tend to disappear by a drying out 

* Paraphrase and direct quotation from Zinsser and Bayne-Jones.i579 



BIOLOGY OF PNEUMOCOCCUS 47 

of colonies after some days, differing in this from streptococcus 
colonies."* 

DIFFERENTIAL MEDIA 

In addition to the diagnostic features of pneumococcal colonies 
grown on Loeffler's medium and on the rabbit blood-agar mixture, 
other media have been recommended as serving to develop charac- 
ters that distinguish pneumococci from streptococci. Hiss/ 50 for 
this purpose, devised a medium consisting of one part of beef se- 
rum and two parts of distilled water, to which was added one per 
cent of inulin (C.P.) and enough litmus to render the medium a 
clear, transparent blue. By fermentation of the inulin, Pneumococ- 
cus acidifies the mixture, causing coagulation of the serum. The 
method is useful except in the case of those rare strains of pneu- 
mococci which are not inulin fermenters. A similar, but solid, me- 
dium for the same purpose was that recommended by Ruediger. 1190 
To sugar-free broth, containing one per cent of Witte peptone and 
1.5 per cent of agar, he added approximately 1.5 per cent of pure 
inulin and a small amount of Merck's highest purity litmus. One 
cubic centimeter of ascites fluid heated to 65° was added to each 
tube of melted agar. Pneumococci are distinguished by the forma- 
tion of red colonies on this medium. 

Buerger 163 claimed that the ring type of colon} 7 on serum, or bet- 
ter serum-glucose agar, was distinctive of Pneumococcus when 
compared with colonies of Streptococcus on the same medium. 
The method required a close inspection of the growth under trans- 
mitted and reflected artificial light. When viewed from the side or 
by transmitted light, the pneumococcal colony shows a distinct 
milky ring enclosing a transparent center, while the streptococcal 
colony has a prominent periphery and a definite nucleus. These 
and other physical characters serve to differentiate these two spe- 
cies of cocci. 

* From Zinsser and Bavne-Jones.i579 



48 BIOLOGY OF PNEUMOCOCCUS 

Bieling, 114 by the use of laked blood agar, laked blood-optochin 
agar, and boiled blood agar, could differentiate pneumococci, and 
the longus and mitior types of streptococci. Presting 1109 used the 
Bieling method in studying some sixty carefully identified strains 
of pneumococci and streptococci, and concluded that boiled blood 
agar was a suitable aid in differentiating pneumococci and green- 
growing and hemolytic streptococci. On the laked blood agar the 
hemolytic streptococci exhibited such marked variation that viri- 
dans could not be distinguished from the hemolytic types, nor from 
pneumococci. Koch, 731 after one year's favorable experience with 
Bieling's blood-optochin agar, recommended the medium for the 
differentiation of pneumococci and streptococci. 

The "polytrope" medium of Lange — a special lactose-mannite- 
peptone bouillon — as reported by Haendel and Lange, 585 develops 
a diffuse orange-yellow coloration when pneumococci of long arti- 
ficial cultivation are grown on it, but remains uncolored when the 
strains are cultivated directly from infected animal material. This 
latter fact renders this medium unsuited to the differentiation of 
bacterial species, because streptococcal strains of long cultivation, 
and other bacterial species as well, cause no color change. Dog- 
blood agar plates, according to Sia and Chung, 1270 give colony 
differences sufficiently pronounced to enable one to distinguish 
between colonies of smooth and rough strains of pneumococci. 
For the same purpose, the authors, in a later communication, 1271 
reported that a medium consisting of one per cent dextrose- 
beef-infusion agar with a reaction equal to pH 7.8 containing 
type-specific antipneumococcic serum served to identify the pneu- 
mococcal type strain. In poured plates, the colonies under the sur- 
face, when illuminated from the side on a dark background, showed 
an annular opacity which was claimed to be type-specific. 

Thomson and Thomson, 1400 by means of photomicrographs of 
colonies of pneumococci grown on plasma-testicular agar, claimed 
that it was possible to detect significant species and even type dif- 
ferences. Their excellent photographs repay study. 



BIOLOGY OF PNEUMOCOCCUS 49 

Viability 

The factors already described, along with temperature condi- 
tions, determine not only the speed and mass of growth but the 
span of life of Pneumococcus. In highly buffered fluids such as 
blood, blood serum, or media containing these body tissues, Pneu- 
mococcus can be preserved alive and virulent for long periods of 
time. Arkharow (1892) 17 found that blood from infected mice and 
rabbits retained its virulence if kept in sealed tubes in the dark at 
room temperature. Foa and Bordoni-Uffreduzzi (1888) 462 incu- 
bated the blood of an infected rabbit for twenty-four hours and 
then stored it in the dark in the cold. Vitality was preserved for 
fifty to sixty days. Rymowitsch 1199 accidentally noted that hemo- 
globin in the medium would conserve viability and virulence for an 
equal length of time at 36° to 38°. Yourevitch 1566 proposed a 
method, based on the protective action of blood and tissue, for 
preserving pneumococci in latent culture. Blood is aspirated by a 
pipette and expelled into the bottom of tubes of glucose or serum 
broth, or a whole heart or fragments of clotted rabbit blood may 
be added. After planting, the cultures are hermetically sealed and 
kept in the ice-box. In such cultures both viability and virulence 
remain unimpaired for several months. 

Romer 1155 preserved pneumococci by mixing the heart-blood of a 
rabbit dead from pneumococcal infection with sterile saline solu- 
tion and storing the infectious material in sealed tubes in a cool, 
dark place. He maintained the virulence of the stock material by 
weekly passage through rabbits. 

Washbourn, 1486 dissatisfied with these methods, preserved stock 
cultures as long as fifty days by covering the growth on agar cul- 
tures with a thin layer of rabbit blood. Gilbert and Fournier 513 
kept strains viable for two months by simple inoculation in fluid, 
defibrinated blood. The cocci still showed capsules and were viru- 
lent for mice. Bezancon and Griffon 112 modified Gilbert and Four- 
nier's procedure by injecting a proteose solution in dogs, then 
mixing their blood with ascitic or pleuritic fluids before seeding 



50 BIOLOGY OF PNEUMOCOCCUS 

with pneumococci. Incubated continuously at 37.5°, the pneumo- 
cocci survived for four months. Truche and Cotoni 1422 added two 
volumes of 15 per cent gelatin in physiological salt solution to 
cultures grown at 37.5° in the "T" medium, sealed the tubes, and 
kept them in the ice-box. In this way viable stock cultures could be 
maintained for at least six months. Ungermann 5435 cultivated pneu- 
mococci in concentrated rabbit serum and then protected the cul- 
tures with a layer of sterile paraffin oil, with incubation at 37.5°. 
Later, Truche 1421 modified Ungermann's method by first growing 
strains on blood or serum agar, and then covering the surface 
with formalinized serum. Preservation continued for a year, yield- 
ing virulent material for inoculation. In a semi-fluid medium con- 
sisting of one part of nutrient agar and five parts of sterile ascitic 
or pleuritic fluids, with short incubation and storage at 8° to 10°, 
Wadsworth (1903) 1454 preserved cultures in a viable condition for 
three or more months. In these exudates without agar, the period 
of viability was even greater. A similar medium with the addition 
of glucose was described in 1930 by Velicanoff and Mikhailova. 1450 

Dehydration is an excellent physical method for the preserva- 
tion of cultures of Pneumococcus, particularly in those cases in 
which experiments continued over a long period of time require 
that the characters of the strain be held uniform and constant. 
Nissen (1891) 1013 first applied this principle to the drying of ster- 
ile silk threads saturated with broth and serum-broth cultures, but 
the results were only indicative of the success which was to attend 
Heim's 631 efforts fourteen years later. Similar threads, soaked in 
the blood of a cat dying of pneumococcal septicemia and dried 
over calcium chloride in a desiccator, fourteen and sixteen months 
later yielded in broth a growth of pneumococci possessing a viru- 
lence equal to that of the original material. 

In the year of Nissen's publication, Bordoni-Uffreduzzi, 142 not 
seeking a method of preservation, reported that pneumonic sputum 
when dried on linen rags gave growth after twelve days' exposure 
to direct sunlight, and as late as after fifty-five days of exposure 



BIOLOGY OF PNEUMOCOCCUS 51 

to diffuse daylight. They concluded that the blood and proteins in 
the sputum formed a protective coating for the cocci. Wood 1542 
studied the longevity of Pneumococcus in sputum when subjected 
to varying temperatures. In moist sputum, kept in the dark at 
room temperature, the average life of Pneumococcus is eleven 
days, while at 0° it is, as a rule, thirty-five days. When the spu- 
tum is kept in strong light at room temperature the life span is less 
than five days. In dried sputum Pneumococcus lives, on an aver- 
age, thirty-five days in the dark, thirty days in diffuse daylight, 
and less than four hours in sunlight. In powdered sputum, death is 
a matter of a few hours. Pneumococci in sprayed, moist sputum 
rarely survive for more than an hour, and often die in less time. 
The material upon which the sprayed particles fall has little influ- 
ence on the life of the organism. On the contrary, Emmerich 355 and 
more recently Stillman, 1326 have demonstrated the longevity and 
continued virulence of pneumococci in a dried condition. Stillman* 
recovered viable organisms from pneumonic sputum dried in test 
tubes and exposed for three months or more to diffuse daylight at 
room temperature. Germano, 511 too, found pneumococci resistant 
to drying, but the details of his experiments are lacking. Hintze 646 
kept blood-agar cultures alive for thirty-two months by storage in 
tubes stoppered with cotton plugs. For the purpose of keeping 
pneumococci viable and virulent, Savino and Acuna 1220 placed the 
blood of mice or rabbits infected with Pneumococcus in one arm of 
an H tube with phosphoric anhydride in the other arm and then 
sealed the tube after evacuating the air. 

The thermal death-point for Pneumococcus as given by Stern- 
berg 1321 is 52° for ten minutes. 

For practical purposes, however, such as the maintenance of 
virulent stock cultures or of other infectious pneumococcal mate- 
rial, in addition to refrigeration of inoculated blood in sealed tubes 
and the devices already described, the rapid drying of cultures, 
infected organs, blood, or sputum presents many advantages. 

* Personal communication. 



52 BIOLOGY OF PNEUMOCOCCUS 

Swift's 1370 method is a successful example. The culture is scraped 
from the surface of the solid medium, spread in thin layers on 
plates or in tubes, and quickly frozen. The frozen material is then 
placed in a chilled Hempel desiccator containing phosphorus pent- 
oxide in the bottom and sulfuric ether in the moat. The drawing 
of a high vacuum dissipates the frozen moisture, and the resulting 
dry powder, if kept anhydrous, is found to contain viable organ- 
isms after many months of storage. Later, Otten, 1040 using a simi- 
lar method for drying thick suspensions of pneumococci in salt 
solution, reported their recovery in living condition after long pe- 
riods of preservation. Desiccated cultures, unlike those on moist 
media, can be shipped over distances involving many days' expo- 
sure to wide temperature variations. 

The reader who desires further details concerning the preserva- 
tion of bacterial cultures is referred to the communication of Flos- 
dorf and Mudd 454 who, after reviewing the literature on the sub- 
ject, described an apparatus for the rapid drying and preservation 
of bacteria, serum, and other biological substances. The method 
consists in applying a high vacuum to the material to be dried 
rapidly and freezing it by immersion in a bath chilled to — 78° by 
the use of Dry-Ice and Methyl Cellusolve. 

Another practical method for having virulent cultures at hand 
is that devised by Neufeld 1000 based on the principle earlier estab- 
lished by Heim. Pieces of spleen or heart or the whole organs of 
mice dead of experimental pneumococcal infection are placed in 
open Petri dishes or in small tubes loosely plugged with cotton and 
dried in a vacuum desiccator over calcium chloride or concen- 
trated sulfuric acid. To recover the pneumococci, generous pieces 
of the dried organs are ground in a mortar with broth, and the sus- 
pension injected intraperitoneally into a mouse. The heart's blood 
of the dead or dying mouse usually yields a pure, virulent culture. 

Autolysis 
Pneumococcus is a delicate organism and may rapidly disinte- 



BIOLOGY OF PNEUMOCOCCUS 53 

grate under certain physical conditions of the medium which favor 
the action of its own lytic ferments. Burgers 187 was among the first 
to study the self-dissolution of pneumococci. He reported that 
autolysis, while favored by the addition of chloroform, was pre- 
vented by heating the organisms to 60° or above. One of the envi- 
ronmental conditions which influence dissolution is the degree of 
acidity of the substrate. Mair 552 gave the range of hydrogen ion 
concentration within which autolysis occurs as from pH 8.5 to 6.0, 
with an optimum of pH 6.8. Lord and Nye 829 observed the dissolu- 
tion of suspended living pneumococci of Types I and II in isotonic 
standard solutions and in approximately isotonic bouillon having 
reactions between pH 4.0 and pH 8.0. Lysis took place with a reac- 
tion higher than pH 5.0 and was most marked in the range pH 5.0 
to 7.0. It was still observable on the alkaline side, but was absent 
when the acidity was greater than pH 5.0. This phenomenon takes 
place at ice-box, room, and incubator temperatures. Mair found 
the rate of lysis increased to a maximum at 42°, and stated that 
this maximum was due to partial destruction of the autolytic fer- 
ment at higher temperatures, and also that the autolysin was more 
sensitive to heat when the reaction was alkaline. Exposure of pneu- 
mococci to 47° for one hour diminishes the degree of dissolution, 
while an exposure of thirty minutes at 56° or five minutes at 100° 
completely arrests the action. Inasmuch as Sternberg 1321 deter- 
mined the thermal death-point as 52° for ten minutes, if the obser- 
vation was correct, the lower temperature should suffice to prevent 
self-lysis, but such is not the case. 

The addition of fresh human serum to suspensions of the cocci 
at varying reactions prevents autolysis. Solutions of lysed pneu- 
mococci added to fresh suspensions of pneumococci in standard so- 
lutions of the same hydrogen ion concentration increase autolysis. 
Cultures of S. viridans, S. haemolyticus, and Staphylococcus 
aureus do not undergo dissolution under similar conditions. Ex- 
tracts prepared by treating minced pneumonic lung tissue with 
chloroform, toluene, and saline solution, or the sediment from such 



54 BIOLOGY OF PNEUMOCOCCUS 

extractions, when added to pneumococci at pH 5.5 to 6.95, dis- 
solve the cells, but no action takes place if the reaction is as acid 
as pH 4.5. From this observation Lord and Nye concluded: "An 
enzyme derived from the bacteria themselves or from the cellular 
material may be the cause of the dissolution." Avery and Cullen 38 " 41 
supported this hypothesis. Autolysates and sterile solutions of 
pneumococci, when added to suspensions of pneumococcal cells in 
phosphate solutions of known pH previously heated to 60° for 
thirty minutes, or 120° for twenty minutes, caused lysis of dead 
pneumococci and, to a less extent, disintegration of closely allied 
organisms such as S. viridans, but they had no effect on Staphylo- 
coccus aureus. The enzyme was most active in a substrate with a 
reaction between pH 6 and pH 8 ; it was destroyed by heating for 
thirty minutes at 60° and was not type-specific in its action. The 
bacteriolytic action was proportional to the concentration of en- 
zyme. Avery and Cullen expressed doubt whether lysis of pneumo- 
cocci under these circumstances was the result of a single enzyme 
or the product of the interaction of more than one. The matter was 
left undecided by their saying that "whether the enzyme or group 
of enzymes concerned in autolysis of pneumococci play any part 
in this form of lysis are questions at present undecided." 
In 1929, Goebel and Avery 520 were able to state that: 

1) Autolysis of Pneumococcus is accompanied by proteolysis, which 
results in an increase in amino and non-coagulable nitrogen; 2) autoly- 
sis of Pneumococcus is accompanied by lipolysis during which there 
is a liberation of ether-soluble fatty acids; 3) when extracts containing 
the active intracellular enzymes are added to heat-killed pneumococci, 
lysis of the cell occurs and there is an increase in the non-coagulable 
and amino nitrogens, comparable to the changes accompanying spon- 
taneous autolysis; 4) when extracts containing the active intracellular 
enzymes are added to emulsions of the alcohol-soluble lipoids extracted 
from pneumococci an increase in the ether-soluble fatty acid occurs. 

In spite of the earlier belief of Jobling and Strouse (1913) 680 
that lysis of pneumococci may be independent of ferment action 
and the statement of Pauli 1071 that this autolysis is due to the self- 



BIOLOGY OF PNEUMOCOCCUS 55 

production of peroxide, it would now seem wise to conclude that 
the self-dissolution of this organism is caused by the action of its 
own intracellular ferments. 

Bile Solubility 

The biochemical reaction taking place in the autolysis of pneu- 
mococci is also involved in the solvent action of bile on the pneumo- 
coccal cell. It was Neufeld 972 who, in 1900, first discovered that 
bile possessed this unusual property, a property which became of 
great diagnostic value in differentiating Pneumococcus from 
Streptococcus and other organisms, and which has since been 
known as the "Neufeld phenomenon." He found that rabbit bile in 
a ratio of 1 to 50, or often of 1 to 200 or 300, added to fresh 
bouillon cultures of living pneumococci, within a fairly wide range 
of temperature of the fluids, rendered them clear and transparent 
in the space of a few minutes. The microscope failed to reveal any 
formed elements, for there was rapid and complete dissolution of 
the pneumococcal cells. The bile of other animal species and of 
man, despite considerable variation in the content of bile-acid salts 
and mucus, exhibited the same property. Bile from rabbits was the 
most active, and clear, limpid bile was to be preferred to that 
which was more viscous or cloudy. Its action was not altered by 
previous heating, but no lysis took place if the cocci had first been 
killed by heat. Neufeld thought this action was attributable to 
cholic acid in combination with glycocol and taurin, since glycocol 
and taurin alone are inactive. 

Nicolle and Adil-Bey (1907) 1007 preferred rabbit bile to that of 
the ox because of its greater solvent action on pneumococci, but 
better still were sodium cholate and sodium choleate. The authors 
found that this property was shared, although to a somewhat less 
degree, by quinolate, taurocholate, glycocholate, and least of all, 
hypocholate. Bile-solubility was looked upon as a salient character 
of Pneumococcus. 

The advantages of sodium taurocholate over whole bile, claimed 



56 BIOLOGY OF PNEUMOCOCCUS 

by Levy, 803 were denied by Mandelbaum, 865 who maintained that 
while the former dissolved strains of Pneumococcus and Strepto- 
coccus mucosus as against a negative action on other streptococci, 
dissolution was not so complete as that produced by bile. The par- 
tial failure of sodium taurocholate may have been due, as pointed 
out by Levy, to lack of uniformity in commercial preparations of 
the salt. A pure preparation of this bile-salt is to be preferred to 
whole bile, because solutions of constant concentration may be 
prepared, sterilized, and kept in stock for quantitative or repeated 
tests. 

Malone 861 would further refine the test by always using the same 
strength of sodium taurocholate (10 per cent), keeping the den- 
sity of the bacterial suspension uniform, employing cultures from 
solid rather than from liquid media, holding both time and tem- 
perature constant, and taking precautions that the mixtures are 
always alkaline in reaction. For routine identification tests such 
refinements seem to be superfluous. 

Neufeld at first held the view that only freshly isolated and viru- 
lent strains of pneumococci were susceptible to the solvent action 
of bile, but Levy, 833 using sodium taurocholate in a 5 to 10 per 
cent solution, believed that the method afforded a clear-cut differ- 
entiation between all pneumococci and streptococci and other bac- 
terial species. It was Levy who, in 1907, with this confirmatory 
test, definitely established Streptococcus mucosus as a pneumo- 
coccus, and called it Pneumococcus mucosus. 

Neufeld's view concerning the correlation between virulence and 
bile-solubility was upheld by Truche, Cotoni, and Raphael 1425 who, 
after an experience of several years, reported that high virulence 
always accompanied complete solubility and total avirulence went 
with insolubility, whereas strains of slight virulence showed partial 
or varying solubility. All thirty-one strains of streptococci tested 
were insoluble in bile. Malone 861 " 2 also observed that pathogenic 
strains and members of fixed types tended to fall into a sodium 
taurocholate soluble group, while the so-called "normal" strains 



BIOLOGY OF PNEUMOCOCCUS 57 

and cocci of Group IV fell into either soluble or insoluble groups, 
with some strains intermediate between the two. 

Schiemann's* observations did not agree with those of Neufeld. 
He found completely avirulent strains which were still bile-soluble, 
as well as mouse-virulent strains which were insoluble. Cocci in the 
blood and peritoneal exudate of freshly dead mice resisted dissolu- 
tion, while transitional forms were only incompletely dissolved. 
Cotoni* described some virulent, typical pneumococci as insolu- 
ble. In marked contrast to these results were those of Falk and 
Jacobson 381 who could find no relation between virulence and bile- 
solubility, since avirulent strains appeared to be as sensitive to the 
lytic action of bile as were virulent cultures. Kelly, 702 too, noted a 
variation in the susceptibility of different strains to lysis, but 
added that specimens of bile differed in solvent power, and that the 
presence of sugar in the medium containing the pneumococci had 
an inhibitory action on the lytic action of bile. Human blood se- 
rum, on the contrary, according to Ziegler, 1570 does not interfere 
with this power. 

Differing in their experience from that of Neufeld, Cole and his 
colleagues 36 found all of several hundred strains of Pneumococcus 
isolated from lobar pneumonia to be bile-soluble. There are rare 
exceptions to this rule, an example being the strain of Pneumococ- 
cus mucosus reported by Dochez and Gillespie. 322 It seems safe to 
say, with Mair, 552 that "different strains of Pneumococcus show 
varying sensitiveness to the action of bile, just as they vary in the 
readiness with which they undergo autolysis in culture, but with a 
satisfactory technique one is very seldom in doubt as to whether a 
particular strain should be classed as bile-soluble or not, and 
strains which have been kept on culture media for long periods re- 
tain the property ."f 

* Quoted by Neufeld and Schnitzer. 

f Sellardsi25* also stressed the importance of the characters of the cultures 
and the alkalinity of the medium in solubility tests. In place of bile Sellards 
substituted 0.01 N to 0.2N solutions of sodium hydroxide, which even in the 
stronger concentration failed to dissolve streptococci. He thought that old 
strains of pneumococci were more soluble than young strains. 



58 BIOLOGY OF PNEUMOCOCCUS 

Harkins 590 contributed the interesting but unexplained observa- 
tion that the bile of animals in which an artificial cholecystitis had 
been set up by intracystic injections of organisms other than 
Pneumococcus (B. coli communior excepted), was devoid of any 
lytic ability. The absence of this property was not due to absorp- 
tion as shown by the fact that the addition of the infecting bac- 
teria (S. mitts, Staphylococcus aureus, and B. coli communior) to 
normal bile failed to rob it of this lytic action. 

Kozlowski 751 further elucidated the action of bile-solubility by 
demonstrating that the higher unsaturated fatty acids in bile were 
even more powerful than cholic acid. The sodium salt of one of 
these fatty acids with an iodine number of 174, in dilutions of ap- 
proximately 1 to 50,000, inhibited the growth of pneumococci and, 
in a dilution of 1 to 5,000, completely killed and dissolved the cells. 
The soaps were active in higher dilutions and in a shorter time 
than were the corresponding salts of taurocholic and glycocholic 
acids. Ziegler 1571 reported that the cytolytic action of sodium de- 
hydrocholate was comparable to the action of sodium taurocholate. 

Downie, Stent, and White 327 tested the solvent action of sapo- 
nin, cholic, dehydrocholic, dehydro-oxycholic, apocholic, and 
desoxycholic acids, as well as a group of choleic acids which are 
addition compounds of desoxycholic acid. Of the substances tested, 
saponin and the sodium salts of all the bile-acids except dehydro- 
cholic and dehydrodesoxycholic acids, brought about lysis. The 
similarity of the action of the sodium salts of the various choleic 
acids to that of sodium desoxycholate indicated to the authors 
that the former owed their activity to the desoxycholic fraction. 

Ziegler 1572 found that sodium dehydrocholate and dehydrodes- 
oxycholate would dissolve pneumococci, thus disagreeing with 
Downie, Stent, and White, who used cultures grown on artificial 
media. Ziegler employed strains obtained directly from infected 
animals without any intervening cultivation. He candidly remarked 
that his paper created more problems than it solved. 

In studying the combined action of alkaline oleates and lino- 



BIOLOGY OF PNEUMOCOCCUS 59 

leates and immune serum on the lysis of pneumococci, Lamar 778 
noted that the oleates rendered the cocci more susceptible to serum 
lysis. In normal serum their action was incomplete, but in immune 
serum there was no multiplication of the organism and lysis was 
complete. As an explanation, Lamar suggested that the action of 
the soap was exerted upon the lipoidal portion of the bacterial cell, 
through which it was rendered more pervious to serum constitu- 
ents and brought under their deleterious and dissolving influence. 

In another paper, Lamar 774 reported that sodium linoleate and 
sodium linolenate killed and dissolved pneumococci more rapidly 
and in higher dilutions than did sodium oleate. Furthermore, in the 
experiments it was seen that blood serum inhibited the bacteriolytic 
action of the unsaturated soaps, partly or completely, depend- 
ing upon the quantitative ratio between serum and soap. Lamar 
concluded that the action was probably caused in part by the 
avidity of the unsaturated fatty acids for protein and not wholly 
by their ability to dissolve lipids. 

In 1926, Falk and Yang 385 compared the solvent action of so- 
dium oleate, sodium hydroxide, tribasic sodium phosphate, and 
saponin on washed pneumococci, and learnt that all these sub- 
stances in the concentrations tested, with the exception of saponin, 
were as specific in their action as is bile. 

In another communication, Falk and Yang 384 described the influ- 
ence exerted by certain electrolytes and non-electrolytes on bile- 
solubility. Chlorides with monovalent cations when present in 
relatively low concentrations inhibited the solution of washed pneu- 
mococci by bile, but in higher concentrations possibly accelerated 
solution. Chlorides with divalent cations acted in reverse fashion, 
inhibiting more effectively in high than in low concentrations. Of 
the anion series tested, sodium hydroxide and tribasic sodium 
phosphate were cytolytic to pneumococci, whereas dibasic and acid 
sodium phosphate, sodium sulfate, and sodium nitrate were not. 
Peptone, gelatin, and ovalbumin appeared to inhibit cytolysis by 
bile in the same manner as calcium or barium chloride. Falk and 



60 BIOLOGY OF PNEUMOCOCCUS 

Yang's observation on saponin does not agree with that of Downie, 
Stent, and White. 

Klein and Stone 721 made the interesting observation that while 
pneumococci were not dissolved by saponin when tested in plain 
broth culture, preliminary treatment of the cocci with cholesterol 
rendered them susceptible to complete and rapid lysis by this sub- 
stance. An excess of saponin inhibited the sensitization by choles- 
terol and, conversely, an excess of cholesterol inhibited lysis by 
saponin. The authors thought, on apparently good evidence, that 
the action of such body fluids as blood and ascitic and pleural 
fluids in sensitizing pneumococci to saponin lysis was attributable 
to the cholesterol content. Klein 720 later reported that ergosterol 
effected a similar sensitization. 

In 1930, Neufeld and Etinger-Tulczynska 983 explained more de- 
tails of the process of bile-lysis. Using bile, sodium taurocholate, 
and sodium linolenate, it was found that bile and bile-acid salts 
possessed the greatest solvent action for pneumococci, although 
soap and bile action were not always parallel. It was further ob- 
served that the same antiseptics (one per cent phenol, one per cent 
formalin, weak ammonium sulfide, acetone, and 0.05 per cent mer- 
curic chloride), which interfere with bile-solubility, in general also 
inhibited spontaneous autolysis as well as the solution of heat- 
killed pneumococci by the bacteriolytic enzyme shown by Avery 
and Cullen 41 to exist in Pneumococcus. Neufeld and Etinger- 
Tulczynska stated that "the effect of antiseptics would indicate 
that bile-solubility and action of autolytic ferments are related," 
but opposed to the concept of parallel bile-solubility and autoly- 
sis there is still evidence that the bile-salts have a lytic action of 
their own, independent of autolytic ferments. The evidence is fur- 
nished by the study of Goebel and Avery 520 on autolysis of Pneu- 
mococcus. In addition to finding that autolysis was accompanied 
by definite proteolysis and lipolysis, they observed that while so- 
dium desoxycholate in excess inhibited the action of pneumococcal 
protease, it did not inhibit the action of the lipase. Goebel and 



BIOLOGY OF PNEUMOCOCCUS 61 

Avery found, furthermore, that when suspensions of pneumococci 
were cooled at 0° — a temperature at which the rate of enzyme ac- 
tion is greatly retarded — the organisms went into solution rapidly 
when sodium desoxycholate was added, and the process was not ac- 
companied by lipolysis or proteolysis. These authors concluded 
that it did not seem probable, therefore, that the bile solution of 
Pneumococcus was identical with the phenomenon of autolysis as 
ordinarily understood and measured. Unpublished work by Dubos 
would, on the contrary, seem to indicate that bile solution occurs 
only under conditions when cell ferments remain active. 

There can be no doubt, however, that the intracellular enzymes 
may act as adjuvants in cell dissolution. The action of extracts of 
living pneumococci in dissolving dead pneumococci (Avery and 
Cullen 41 ) with an increase in non-coagulable and amino nitrogen 
and in ether-soluble fatty acids (Goebel and Avery 520 ) favor this 
view, a view which has received additional support by the experi- 
ments of Wollman and Averbusch, 1535 " 36 who were able to cause 
bile solution of killed non-soluble, virulent, and avirulent pneumo- 
cocci by the addition of a small amount of a living, avirulent cul- 
ture to the bile-culture mixture. This phenomenon they called 
autolyse transmissible. 

Atkin 29 made the original observation that organisms compris- 
ing the papillae which frequently arise as secondary growths on 
autolyzed colonies of pneumococci on serum agar were quite in- 
soluble in bile. It was evident that the organisms of which the 
papillae consist were devoid of autolysin since they showed no 
tendency to undergo dissolution or self-digestion even after several 
weeks of incubation. The organisms thus derived, when subcultured 
on fresh serum agar, regained autolytic power and at the same 
time became bile-soluble. Atkin made the statement that "bile solu- 
bility of pneumococcus is due to an acceleration of the normal 
autolytic process by this substance, and that no solution of the or- 
ganism occurs except in the presence of the autolysin." 

Another observation which contributed to the question was that 



62 BIOLOGY OF PNEUMOCOCCUS 

of Lord and Nye, 835 " 7 who found that more rapid dissolution of 
pneumococci occurred in bile than in standard buffer solutions and 
that lysis proceeded at a more rapid rate when the reaction of the 
mixture was slightly alkaline. The authors attributed the effect to 
the more rapid death of the organisms in bile and, hence, to the 
liberation of a larger amount of enzyme at the optimal hydrogen 
ion concentration. 

Whatever may be the facts regarding the exact nature of bile 
solution of Pneumococcus, it cannot be gainsaid that this phe- 
nomenon and that of autolysis bear striking similarities. Whether 
or not they are due to entirely separate mechanisms, bile may be 
regarded as an accelerator of natural autolysis. 

Sensitiveness to Germicides and Other Chemical Substances 
Pneumococcus is highly vulnerable to the usual antiseptics and 
germicides. The problem of killing pneumococci and of rendering 
infectious material harmless is a simple one, and can be dismissed 
with scant discussion. As an example of the effective concentra- 
tions of two common germicides, Schiemann and Ishiwara 1230 gave 
for the growth-inhibiting strength of phenol and mercuric chloride 
1 to 600 and 1 to 100,000, respectively. These figures were ob- 
tained by adding a small quantity of a twenty-four-hour broth 
culture of pneumococci to 5 per cent ox-serum containing varying 
dilutions of the two substances.* 

Simon and Wood 1288 " 9 investigated the action of a large number of 
dyes and concluded that the majority of the basic type, especially 
those of the triphenylmethane series, in a concentration of 1 : 100,000 in 
agar were inhibitory for pneumococci as tested by making a stroke cul- 
ture on the medium. Acriflavine (trypaflavine) acts powerfully both on 
virulent and non-virulent pneumococci (Schiemann and Baumgarten, 
1923). Norton and Davis (1923) found that organisms belonging to the 
S. viridans and Pneumococcus groups were inhibited by dyes to the 
same extent, so that none could be used for the purpose of differentiat- 
ing between them. 

* Quoted from Browning.i 5 ^ 



BIOLOGY OF PNEUMOCOCCUS 63 

For killing cultures to be used as antigens for the production of 
active immunity 0.5 per cent phenol and 0.3 per cent formalin are 
commonly used. Sodium ricinoleate was first used for this purpose 
by Larson and Nelson, 791 because of its rapid lethal effect on pneu- 
mococci and the absence of any impairing action on the antige- 
nicity of the treated cells. Barnes and Clarke 81 determined that 
sodium ricinoleate and sodium oleate were pneumococcidal in con- 
centrations of 0.04 and 0.004 per cent respectively, which, in con- 
junction with Lamar's results on the action of sodium lanolate, 
give us definite figures for these soaps. 

In discussing the effect of various chemical substances on the vi- 
tality of Pneumococcus, mention may be made of the method devel- 
oped by Schnabel 1238 " 9 for measuring such effects. Based on the re- 
ducing power of pneumococci, he determined the ability of the 
organism in contact with varying strengths of the test chemicals 
to produce significant color changes in methylene blue, litmus, neu- 
tral red, indigo blue, and other dyes. Since the reducing power of 
pneumococci is a variable one, this factor must be carefully con- 
trolled by proper observance of time, temperature, and hydrogen 
ion concentration. The method enabled Schnabel to study both the 
inhibiting and the sensitizing action of quinine, optochin, mercuric 
chloride, formaldehyde, phenol, and silver nitrate on Pneumococ- 
cus. The method holds possibilities for the study of drug-fastness 
of the coccus and Schnabel claimed that by the use of this tech- 
nique he could determine the presence of optochin in a 1 to 1,000,- 
000 dilution and that it can be applied to the quantitative meas- 
urement of the amounts of analogous substances in solution. 

The discovery by Morgenroth and Levy 926 that quinine and some 
of its derivatives, especially optochin, possess bactericidal powers 
for Pneumococcus, both in vitro and in vivo, has led to investiga- 
tions on the therapeutic effect of this class of substances on pneu- 
mococcal infection. The results of the studies will be discussed in 
the chapter on Chemotherapy. 

To summarize our knowledge of the bactericidal action of vari- 



64 BIOLOGY OF PNEUMOCOCCUS 

ous chemicals upon Pneumococcus, it suffices to say that no sub- 
stance or class of substances have as yet been found which exert 
any selective lethal action on the cell with the exception of bile- 
acids and certain derivatives of quinine. 



CHAPTER III 
BIOCHEMICAL FEATURES 

The behavior of Pneumococcus toward proteins, fats, and carbo- 
hydrates; acid production; oxidizing and reducing action; and 
the formation of peroxide, methemoglobin, hemolysin, hemotoxin, 
and alleged endotoxin. 

The pneumococcal cell is charged with a complement of en- 
zymes capable of destroying it as well as certain extracel- 
lular substances upon which they are allowed to act. The living or- 
ganism and sterile extracts made from it can digest proteins, split 
lipids, and invert and ferment carbohydrates. 

Proteolysis, Lipolysis, and Carbohydrate Fermentation 

PROTEOLYSIS 

Rosenow 1169 was probably the first to express the opinion, based 
on experimental evidence, that Pneumococcus contained a proteo- 
lytic enzyme capable of splitting its inherent protein into a highly 
poisonous substance and to demonstrate the proteolytic action of 
Pneumococcus. Extracts of virulent pneumococci made in sodium 
chloride solution and filtrates of broth cultures hydrolyzed the 
proteins contained in heated ascites-meat broth and, to a lesser 
degree, those of heated serum. The extracts did not attack egg- 
white or pure casein. The enzyme appeared to be more resistant to 
heat and to long standing in broth filtrates than in salt solution. 
Heating at 60° reduced the enzymatic action of the filtrates by 50 
per cent, and almost completely destroyed the activity of the saline 
extracts. 

In 1920, Avery and Cullen 38 gave a still more detailed demon- 
stration of the proteolytic action of Pneumococcus. By dissolving 
living cocci in bile or sodium cholate, or by allowing them to auto- 



66 BIOLOGY OF PNEUMOCOCCUS 

lyze under alternate freezing and thawing in phosphate solutions 
with a reaction of pH 6.2, or by filtering sterile broth cultures, the 
authors obtained cell-free extracts capable of hydrolyzing casein 
and fibrin but not albumin and gelatin. The extracts digested pro- 
teoses and peptones even more rapidly, converting them into pep- 
tides and amino acids. The enzyme or enzymes were designated by 
the authors as protease and peptonase, although it was stated that 
these names might merely represent different activities of the same 
ferment. The activity of the enzyme was favored by a reaction 
of pH 7.0 to 7.8, but was suspended at pH 5.0. The enzyme, 
however, was not destroyed by relatively short exposure to this 
acid reaction, since its activity was restored when the pH of the 
solution was readjusted to 7.8. The rapidity of hydrolysis was 
proportional to the concentration of enzyme. Heating diminished 
the digestive action, which was lost after ten minutes' exposure to 
100°. The ability of active extracts to digest the protein of heat- 
killed pneumococci was later shown by Goebel and Avery 520 by 
demonstrating an increase in the non-coagulable and amino nitro- 
gen after contact of the extract with the killed cocci. 

LIPOLYSIS 

The presence of an esterase in the pneumococcal cell has also 
been demonstrated by Avery and Cullen. 39 Extracts of pneumo- 
cocci, prepared in the manner employed for obtaining proteases, 
split tributyrin. Heating for ten minutes at 70° destroyed the 
activity of the lipase. While bile and bile-salts accelerated lipoly- 
sis, they were not essential to the reaction, since pneumococcal ex- 
tracts prepared in other ways exhibited the same enzymatic ac- 
tivity. In the study of Goebel and Avery it appeared that on the 
addition of extracts containing the active intracellular enzymes of 
the pneumococcal cell to the ether-soluble lipids of pneumococci, 
an increase in ether-soluble fatty acid occurred. Falk and Mc- 
Guire 386 recently reported that strains of Pneumococcus, Types I 
and II, in suitable broth, hydrolyzed phenylacetate, glyceryl tri- 



BIOCHEMICAL FEATURES 67 

acetate, methyl n-butyrate, and benzylacetate. While different de- 
grees of hydrolysis were shown for the four esters, the comparative 
ratio of the action of the two types of organisms on these esters 
was of the same order in different lots of broth, although the abso- 
lute values were not the same. As a rule, Type I organisms showed 
a greater action than those of Type II, except in the case of 
phenylacetate. 

CARBOHYDRATE FERMENTATION 

Important from a practical standpoint is the content in Pneu- 
mococcus of carbohydrate-splitting enzymes, since the selective ac- 
tion of the latter serves in the bacteriological differentiation of 
this species from other species and genera of the family Coccaceae. 

Invertase, amylase, and inulase were first demonstrated in Pneu- 
mococcus by Avery and Cullen (1920). 40 Finding that bile solu- 
tions of pneumococci were unsuited to the purpose, the authors 
used sterile extracts made by alternately freezing and thawing sus- 
pensions of cells in balanced phosphate solutions at a pH of 6.2. 
The enzymes were active within the limits pH 5.0 to 8.0, with an 
optimum of 7.0. The acid death-point of pH 5.0 may be reached in 
carbohydrate media, even when buffered with phosphates, if the 
content of glucose exceeds 0.3 per cent (Avery and Cullen, Lord 
and Nye 829 ). These authors agreed in ascribing carbohydrate fer- 
mentations to intracellular or endoenzymes. The saccharolytic fer- 
ments of Pneumococcus have a lower thermal death-point than the 
protease or lipase, being destroyed at 55° in ten minutes. 

The enzymatic preparations freed of living cells hydrolyze su- 
crose, starch, and inulin but, strange to say, fail to ferment glu- 
cose, although in culture media the cleavage of this sugar into acid 
is a definite and constant activity of the growth of Pneumococcus. 
According to Hewitt, 643 the breakdown of glucose by Pneumo- 
coccus is a less complex process than in the case of many other 
organisms. Of the glucose constantly disappearing from such cul- 
tures, about 78 per cent was recovered by Hewitt as lactic acid. 



68 BIOLOGY OF PNEUMOCOCCUS 

There appeared to be marked and characteristic divergences in the 
behavior of different types of pneumococci toward this sugar, the 
variant RIII being first and SI second in order of glycolytic ac- 
tivity. It was Hewitt's belief that inorganic phosphorus played an 
essential part in the fermentation of glucose and that this part 
was independent of its buffering action. The full benefit to Pneu- 
mocQecus from phosphates was only obtained when media contain- 
ing these salts were sterilized by filtration and not by autoclaving. 

The cleavage of maltose and lactose, unlike that of glucose, is 
due respectively to a maltase and a lactase in the pneumococcal 
cell (Fleming and Neill 402 ), while Neill and Avery 954 have demon- 
strated a raffinase. 

A significant and distinctive enzymatic property of Pneumococ- 
cus is its ability to ferment inulin, a polyose from dahlia bulbs. It 
was Hiss 649 who first in 1902 discovered that, by means of this 
property, it was possible to differentiate pneumococci from the 
closely related members of the tribe Streptococcaceae. Serum- 
water containing one per cent inulin supported growth of Pneumo- 
coccus, resulting in the development of acidity and coagulation, 
while streptococci, although growing, failed to form appreciable 
acid or to coagulate the medium. It was the use of this method 
which, with other biological characters, indicated that the so-called 
Streptococcus mucosus was in reality a pneumococcus. 

Duval and Lewis 342 overcame the variations encountered in va- 
rious batches of serum-water by using inulin broth. They found 
that in its fermentative ability Streptococcus mucosus closely re- 
sembled Pneumococcus, but decided that the inability to ferment 
inulin did not necessarily exclude an organism from the species, 
since some pneumococci failed to show this character. Dochez and 
Gillespie 822 described a strain of Pneumococcus mucosus that did 
not ferment inulin. Berry 106 also observed the pneumococcal char- 
acters of S. mucosus, and her results agreed with those of Duval 
and Lewis in that she encountered other strains of pneumococci 
that failed to attack inulin, as well as variations in this respect 



BIOCHEMICAL FEATURES 69 

in strains that had undergone changes in morphology and viru- 
lence induced by longer or shorter cultivation on artificial media. 
Berry drew attention to the lack of uniformity in the serum-water 
medium and in commercial preparations of inulin. With a reliable 
lot of inulin serum-water, however, some non-inulin-fermenting 
strains acquired or regained this fermentative property after 
animal passage. 

In a more recent paper, Berger and Silberstein 103 reported the 
results of a study on strains of pneumococci, of the "B" modifica- 
tion* of this species, and of green-producing and hemolytic strep- 
tococci. The hemolytic streptococci; none of which affected inulin, 
were the only group with constant behavior toward this carbo- 
hydrate. Of the typical pneumococci, four strains showed redden- 
ing without coagulation of the medium and two had no inulin-fer- 
menting power. These two strains were found to be modification 
"A"* when tested with optochin. Of nine strains of modification 
"B" obtained from Pneumococcus, but behaving otherwise as green 
streptococci, two retained the inulin-fermenting property. Of 
thirty green streptococci, five gave marked inulin fermentation, 
while four others showed slightly positive reactions. Other authors 
to report strains incapable of fermenting inulin among the pneu- 
mococci are Hiss ; Park and Williams ; Levy ; Avery ; and Burger 
and Ryttenberger.f 

In addition to inulin, the polysaccharide, glycogen, is equally 
susceptible to the saccharolytic action of Pneumococcus. First re- 
ported by Hiss, 649 this reaction has been quantitatively studied by 
Barnes and White, 85 who compared the fermentative reactions of 
strains of Type I, II, III, and V pneumococci on glucose, inulin, 
mammalian glycogen, and glycogen obtained from scallops. The 
authors decided that, on the whole, the four type strains used fer- 
mented glycogen to essentially the same degree as they did glucose 
and inulin. 

Pneumococcus attacks still other carbohydrates, but no specific 

* To be described in Chapter V. f Quoted by Neufeld and Sehnitzer. 



70 BIOLOGY OF PNEUMOCOCCUS 

enzymes have been demonstrated for these reactions. In addition 
to glucose, galactose and levulose are actively fermented with the 
formation of lactic acid, as well as the trisaccharide, trehalose, 
and the glucoside, salicin. The fermentation of alcohols is much 
less marked. Slow acid production takes place in media containing 
glycol, glycerol, and erythritol; some strains slowly produce acid 
from mannitol, but there is no action on dulcitol or sorbitol. The 
pentoses, arabinose and xylose, are slowly attacked (Mair 552 ). Zo- 
zaya 1586 added levan, obtained from B. mesentericus and B. sub- 
tilis, to the list of substances fermentable by Pneumococcus. Dex- 
tran, obtained from Leuconostoc mesenteroides was not affected. 
Although the change in levan was less than that in inulin, the 
acidity in both cases seemed to be mainly due to lactic acid. 

Acid Production 

Supplementing the brief mention that has already been made of 
the formation of acid from saccharides by Pneumococcus, some of 
the factors that condition that reaction are as follows: The 
amount of sugar present in the medium determines the degree of 
acidity attained. If sufficient carbohydrate is present, growth 
ceases at an acid reaction of pH 5.0. If there is less than 0.4 per 
cent of sugar, growth ceases at a lower hydrogen ion concentra- 
tion, apparently because of exhaustion of carbohydrate. If no 
carbohydrate is present save that extracted from the meat from 
which the broth is made, growth initiated at pH 7.8 ceases at 
about pH 7.0. If the reaction of bacteria-free filtrates of plain 
broth cultures in which growth has ceased is readjusted to pH 7.8 
and the medium reinoculated with Pneumococcus, no growth oc- 
curs unless carbohydrate is added. However, if bacteria-free fil- 
trates of dextrose-broth cultures in which growth has ceased are 
readjusted to pH 7.8 and reinoculated with Pneumococcus, growth 
is resumed. Cultures of Pneumococcus, with maltose, saccharose, 
lactose, galactose, raffinose, dextrose, and inulin give identical re- 
sults in the rate of reaction change and the final hydrogen ion 



BIOCHEMICAL FEATURES 71 

concentration (pH 5.0) attained. Such are the conclusions of 
Avery and Cullen 37 in their 1919 study. Jones, 6S3 a year later, ex- 
plained the failure of these authors to produce an acidity greater 
than a final pH of 7.0 by the fact that they did not add glucose to 
their beef-infusion medium, but it would seem that this failure had 
already been accounted for in other parts of their report. 

The degree of acid production by pneumococci appeared to 
Gundel 569 to be correlated with pathogenicity. He grew strains 
isolated from human mouths in lactose bouillon (pH 7.0) and in 
this medium noted only a slight acid formation by pathogenic 
strains, whereas non-pathogenic pneumococci produced marked 
acidity with transition forms between the extremes. No confirma- 
tion of Gundel's results appears to have been published. 

The addition of normal human serum to suspensions of living 
pneumococci in isotonic bouillon, while preventing cell dissolution, 
does not inhibit acid production (Lord and Nye 834 ). In rabbit 
serum, according to Bordet, 139 " 40 the pneumococcidal hydrogen ion 
concentration is pH 6.2 for Types I, II, and III, and here the 
acidity is due to formic and acetic acids. 

Oxidation and Reduction 
Pneumococcus, under varying degrees of oxygen tension, mani- 
fests the phenomena of oxidation and reduction. With the activi- 
ties of its oxidases are correlated, either directly or indirectly, 
synthesis and hydrolysis of pneumococcal protein and carbohy- 
drates, changes in the hydrogen ion concentration, the formation 
of peroxide, the conversion of hemoglobin into methemoglobin, the 
lowering of the oxygen capacity of blood cells, and the death of 
the organism itself. 

METHEMOGLOBIN PRODUCTION 

In 1913, Butterfield and Peabody, 194 seeking an explanation for 
the reduction of the 2 capacity of the blood in human lobar pneu- 
monia, discovered that upon incubating pneumococcal cultures 



72 BIOLOGY OF PNEUMOCOCCUS 

with washed rabbit corpuscles, there ensued a diminution of the 
oxygen-carrying capacity of the cells, owing to the formation of 
methemoglobin, or some derivative of hemoglobin with identical op- 
tical constants for these regions of the spectrum. The substance 
which induced this change was also present in sterile filtrates of 
autolyzed cultures. The authors concluded that the mechanism of 
the reduction of the 2 -carrying capacity of the blood in human 
lobar pneumonia was, in part at least, of the same nature. In rab- 
bits with a severe experimental bacteriemia it was found that the 
2 -combining power of the venous blood fell progressively up to 
the time of death. Coincidentally, there was an even more marked 
fall in the 2 content of the arterial blood. The changes in the 
blood of infected animals appeared to be analogous to those seen 
when Pneumococcus was grown in blood in vitro, the oxygen-car- 
rying capacity of the cells in both instances being due to the con- 
version of hemoglobin into methemoglobin. 

In the next year (1914), Cole 253 reported conclusions which, be- 
cause of their importance, may be summarized here: Pneumococci 
in contact with hemoglobin transform this substance into methemo- 
globin, and the reaction occurs only when the pneumococci are liv- 
ing; it is not induced by the culture fluid or by extracts of the 
bacteria, differing in this respect from the results of Butterfield 
and Peabody. The reaction does not occur when hemoglobin is 
added to an emulsion of washed pneumococci in salt solution. How- 
ever, if minute traces of dextrose be added to the mixture, the 
transformation occurs quickly. Dextrose may be replaced by any 
one of a number of other sugars, and also by some organic com- 
pounds, if the latter are added in large amounts. Certain other or- 
ganic substances were unable to replace dextrose, but it was impos- 
sible to determine any special molecular configuration upon which 
this property depends. The formation of methemoglobin by pneu- 
mococci probably resembles the changes induced in the blood by 
such chemical substances as amino phenol. It seems not unlikely 
that the transformation is always a reaction of oxidation. In the 



BIOCHEMICAL FEATURES 73 

presence of reducing agents, the latter are first oxidized, the ac- 
tion occurring more readily in the presence of oxyhemoglobin. In 
some instances an alternate oxidation and reduction of the trans- 
formative agent occurs, so that the reaction is continuous. The ef- 
fect of the presence or absence of free 2 on the changes in blood 
pigment induced by chemical agents of known composition sug- 
gests that the reaction with Pneumococcus follows similar lines. 
The reaction does not occur in the absence of 2 . If the free 2 be 
first removed and then replaced, the reaction takes place more 
rapidly than if the 2 had not been removed. The presence of free 
2 in excess slightly delays the reaction, possibly because of the 
inhibition of the reduction process which forms the first part of 
the reaction. 

In 1924, Morgan and Neill, 916 as a result of their experiments, 
concluded that: 

Sterile filtrates of aerobic cultures of Pneumococci containing H 2 2 , 
were capable of converting catalase-free solutions of oxyhemoglobin 
into methemoglobin. In catalase-containing solutions of hemoglobin 
from laked corpuscles, the actual methemoglobin-forming system of 
Pneumococcus involves a labile constituent of the bacterial cell, which is 
itself susceptible to oxidizing agents and may be rendered inactive if 
exposed to peroxide or similar substances previous to its introduction 
into oxyhemoglobin solutions. The activity of this function, in the case 
of sterile filtrates, depends, therefore, upon the liberation of cell con- 
stituents into the medium and upon the protection of those cellular sub- 
stances from the oxidizing agents which are formed when Pneumococ- 
cus cultures are freely exposed to air. When these cultural conditions 
are fulfilled, sterile culture filtrates of Pneumococcus convert oxyhemo- 
globin into methemoglobin independent of the presence of blood cata- 
lase. 

In 1921, Stadie, 1311 in studying the blood changes in pneumo- 
coccal infections, found that methemoglobin disappeared rapidly 
from the blood stream, whether introduced by injection or formed 
within the circulation by pneumococci or by the action of chemicals 
(potassium ferricyanide and sodium nitrate). He concluded: "In 



74 BIOLOGY OF PNEUMOCOCCUS 

the occasional cases of pneumonia which show a decrease in the 
oxygen capacity of the blood, the decrease is probably due to the 
formation of methemoglobin. The latter is removed from the circu- 
lation, however, as rapidly as it is formed, so that it can seldom be 
detected even qualitatively, and is probably never the cause of 
cyanosis." The practical impossibility of demonstrating methemo- 
globin in the circulating blood was also experienced by Schnabel. 1233 
The explanation of the phenomenon of methemoglobin produc- 
tion is of importance not only in so far as this special reaction is 
concerned, but also because it suggests a mechanism by which 
pathological effects may be produced by bacteria which appar- 
ently elaborate no soluble toxin. 

PEROXIDE FORMATION 

The work of McLeod and Govenlock, 885 of McLeod and Gor- 
don, 881 " 3 and of McLeod, Gordon, and Pyrah 884 was confirmatory 
of Cole's results and amplified our knowledge of the property pos- 
sessed by pneumococci to form peroxide during growth. The au- 
thors ascribed the greenish or yellowish discoloration produced 
on heated blood media to an accumulation of peroxide, showed that 
the death of pneumococci in cultures was brought about by an ex- 
cess of the same product, and demonstrated that the substance was 
not organic peroxide but hydrogen peroxide. It was not formed in 
cultures deprived of oxygen, nor in cultures that contained abun- 
dant catalase. 

Penfold 1077 disclosed another manifestation of the oxidizing ac- 
tion of pneumococci. According to him, all pneumococci and prob- 
ably all streptococci acting on certain aromatic amines, notably 
aniline, benzidine, and the toluidines, produce pigment. When 
pneumococcal and streptococcal cultures were grown on citrated 
horse-blood agar containing benzidine, the colonies appeared black 
by transmitted light and metallic blue by reflected light with dark 
discoloration extending far into the medium around the colony. 
This pigment production from the amines is a peculiarity shared 



BIOCHEMICAL FEATURES 75 

only by pneumococci and streptococci and, according to Penfold, 
is due to the peroxide produced by these organisms. 

In the next year, Felton 395 studied the oxidase reaction of bac- 
teria by means of the oxidation of p-amino leuco-malachite green, 
and found that the conditions most suitable for the reaction were 
the presence of a slight amount of hemoglobin and of dextrose, an 
acid reaction, the presence of fresh serum heated for thirty min- 
utes at 56°, and a plentiful supply of oxygen. In order of decreas- 
ing suitability for promoting oxidation were rat, guinea pig, rab- 
bit, horse, human, cat, and chicken serum. Of the organisms tested 
by Felton, only Pneumococcus, S. viridans, and S. haemolyticus 
exhibited oxidative power, the first named giving the most intense 
reaction. That this power was not an exclusive character of the 
three species was shown in the same year (1923) by McLeod, Gor- 
don, and Pyrah, 884 who added B. bulgaricus and B. acidophilus to 
the group of peroxide-producing bacteria. 

Directly opposed to the conclusions of McLeod and Gordon and 
of Cole, but somewhat analogous to those of Felton, were the views 
of Barnard and Gowen. 80 These two authors discounted the possi- 
bility that the green disintegration product of hemoglobin refer- 
able to the growth of pneumococci is methemoglobin, since methe- 
moglobin will not exist in the presence of hydrogen peroxide. A 
green pigment, identical in all respects with that produced by the 
growth of pneumococci on hemoglobin, was prepared by Barnard 
and Gowen by the action of peroxide and certain nitrogenous com- 
pounds on blood pigment. A chemical study of this artificial green 
pigment indicated to the authors that it was not methemoglobin 
but a xanthoprotein compound. Without more complete informa- 
tion it is impossible to judge the correctness of the claim, but 
studies from the Hospital of the Rockefeller Institute seem to 
prove the case for methemoglobin. It is, however, possible that the 
appearance of xanthoprotein may be an additional effect of 
peroxide. 

Avery and his colleagues published a series of papers that mate- 



76 BIOLOGY OF PNEUMOCOCCUS 

rially amplified our knowledge of this phenomenon of peroxide pro- 
duction by bacteria. Avery and Morgan 52 noted the early appear- 
ance of peroxide in cultures of pneumococci and of non-hemolytic 
streptococci, with a somewhat retarded development in cultures of 
some but not all strains of S. haemolyticus and S. mucosus. Per- 
oxide was not detected at any time during the growth of two 
strains of Staphylococcus aureus. The factors influencing peroxide 
formation were free access of air, and the absence of a catalase, 
peroxidase, or other catalyst capable of decomposing H 2 2 . Under 
favorable conditions peroxide production continued during the 
logarithmic phase of growth and persisted for at least six to twelve 
days. According to Avery and Morgan the aerobic growth of 
anaerobic bacteria in broth containing sterile, unheated plant tis- 
sue may be related to the action of oxidizing-reducing systems of 
plant tissue in the destruction of toxic peroxides formed during 
bacterial growth. These assumptions were supported by subse- 
quent experiments by Avery and Neill, 55 in which it was found that 
anaerobically grown pneumococci rapidly form peroxide on ex- 
posure to molecular oxygen. The peroxide-forming properties of 
pneumococci varied with different strains and with the age of the 
cells, and were active under conditions of reaction and temperature 
that did not permit active cell growth and multiplication — a range 
of pH 5.0 to 8.5 — and at lower temperatures than those that fa- 
vor growth. 

Avery and Neill, 56 employing sterile, filtered extracts prepared 
by freezing and thawing pneumococcal cells or by dissolving them 
in bile, proved that the peroxide-forming ability of Pneumococcus 
is a function not dependent upon the presence of living, intact 
cells. Sterile extracts of unwashed pneumococci promptly formed 
peroxide on exposure to air; its formation was almost as active in 
extracts aerated at 2° as in those exposed to air at room tempera- 
ture. The optimal zone was on the alkaline side of pH 6.0, but 
peroxide was detectable in the range pH 5.0 to 9.0. The peroxide- 



BIOCHEMICAL FEATURES 77 

forming activity of the cells was gradually diminished by pro- 
longed exposure to 55° and was destroyed by heating for five min- 
utes at 65°. Moreover, cells and extracts of cells thoroughly 
washed before extraction with salt or phosphate solution showed 
no peroxide-producing activity, but such extracts could be acti- 
vated by the addition of cell washings, yeast extract, or muscle 
infusion. 

In a subsequent paper, Avery and Neill 57 reported that sterile 
broth extracts of unwashed pneumococci, entirely free from living 
or intact cells, actively reduced methylene blue, whereas sterile ex- 
tracts of washed pneumococci in phosphate buffer solution were 
unable by themselves to reduce the d} T e. As in the case of peroxide 
formation, the reducing action was restored by the addition of 
meat infusion or yeast extract to extracts prepared from washed 
cells. Piatt 1097 reported that meat extract augments the amount of 
peroxide formed as do also lactic acid and glucose. Gelatin, on the 
contrary, delays its formation, and in gelatin broth no appreciable 
amounts of peroxide are present until near the end of the period of 
logarithmic increase of bacteria. 

The system or systems responsible for methylene blue reduction 
are destroyed by exposure to temperatures practically identical 
with those which had previously been found to destroy the per- 
oxide-forming activity of the same extracts. Avery and Neill 57 sug- 
gested that peroxide formation and methylene blue reduction by 
pneumococcal extracts are functions of the same or closely related 
systems, the particular reaction induced depending upon whether 
molecular oxygen or methylene blue serves as hydrogen acceptor 
or oxygen donator. 

In 1930, Hewitt 641 supplied further confirmatory data concern- 
ing the phenomena of oxidation and reduction by pneumococci and 
streptococci. In aerobic cultures of both species there is a rise in 
potential after the logarithmic phase of growth, while in the case 
of diphtheria bacilli and staphylococci, the potential remains at a 



78 BIOLOGY OF PNEUMOCOCCUS 

low level long after the cessation of active cell proliferation. In a 
second paper, Hewitt 642 reported the use of liver, blood, or bac- 
teria in culture media as a means of inhibiting peroxide formation 
and of permitting the study of other oxidation-reduction phe- 
nomena. In aerated cultures under these conditions, the potential 
fell to much lower levels and growth was much more luxuriant. In 
similar cultures without catalase the potential first declined, then 
rose to the level at which peroxide can be detected chemically. In 
the presence of catalase, however, the potential fell, then rose very 
slowly, and finally reached the level corresponding to peroxide ac- 
cumulation only after the effective catalase had been destroyed by 
the active oxidizing system. If fresh catalase was then added an 
immediate drop in potential occurred. 

Lieb 812 did not agree that the oxidation phenomenon had to do 
with the formation of hydrogen peroxide but thought that it was 
due to another mechanism operated by an unknown oxygen-carry- 
ing factor, possibly a ferment, acting in the same oxygen concen- 
tration as hydrogen peroxide. Such a conclusion would appear to 
be no more than conjecture. 

Pauli's work ( 1927-8 ) 1071 was more or less a repetition of that 
of the American school. He explained the fact that Pneumococcus 
rapidly undergoes autolysis in cultures by assuming that lysis was 
due to the production of hydrogen peroxide in the absence of cata- 
lase. When he grew pneumococci, respectively, in the presence of 
air, under an atmospheric pressure of thirty millimeters of mer- 
cury, or under complete anaerobic conditions, the cells grew well, 
but autolysis took place rapidly in the first, slowly in the second, 
and was still absent in the third after a month's incubation. Pauli 
ascribed the advantage of adding any of the many growth-stimu- 
lating substances to culture media to the content of catalase which 
decomposes hydrogen peroxide at the time of its formation. 

Another effect of the oxidizing agents formed when sterile ex- 
tracts of pneumococci are exposed to air is the destructive action 



BIOCHEMICAL FEATURES 79 

they exert on the saccharolytic enzymes, sucrase, raffinase, inulase, 
and amylase, of the cell. Aerated extracts, however, have no inacti- 
vating effect on pneumococcal protease or lipase. 

The relative resistance of the enzymes of Pneumococcus to heat 
is consistent with their resistance to the action of hydrogen per- 
oxide. Neill and Avery 903 found, furthermore, that sterile ex- 
tracts of autolyzed anaerobic cultures of pneumococci contained 
much higher concentrations of active endocellular enzymes than 
did filtrates of autolyzed aerobic cultures. The difference may be 
explained by a destruction of the formed enzymes by oxidative re- 
actions analogous to the destruction observed during oxidation of 
sterile broth extracts of unwashed pneumococcal cells. The more 
complete destruction of enzymatic activity in autolyzed aerobic 
cultures than in oxidized sterile cell extracts is probably due to 
the longer exposure to oxidation products. More recently Finkle 
(1931), 440 in a study of the metabolism of pneumococcal variants, 
confirmed the fact that oxidation may have an inhibitory effect on 
sugar fermentation. Under anaerobic conditions saccharolysis is 
approximately the same for organisms of Types I, II, and III, 
while, under aerobic conditions, only Type I cells are capable of 
this action. The difference may be explained by the relatively 
feebler respiratory capacity of Type I organisms. The capacity of 
these cells is only 56 per cent of that of Type III pneumococci, 
which in turn is 71 per cent of that of Type II strains. 

In a later paper, Neill and Avery 956 reported further details of 
the oxidation-reduction system of Pneumococcus. The thermosta- 
ble components of the oxidation-reduction system were still active 
in oxidized extracts, while the thermolabile cellular constituent was 
destroyed not only by products formed during oxidation, but also 
by molecular oxygen itself. The labile component of the system 
was more susceptible to the action of reagent hydrogen peroxide 
than any other known intracellular substance of pneumococcal ori- 
gin. Neill and Avery designated the thermostable substances as 



80 BIOLOGY OF PNEUMOCOCCUS 

i?H, the actual source of the oxidizing agents, and the thermo- 
labile cellular constituent as C, serving as a catalyst in acceler- 
ating the reaction, which they formulated thus : 

1) C + -RH -\- 2 — » oxidizing agent 

2) Oxidizing agent -f- C — > inactive C. 

i?H then represents substances that are not present in Pneumo- 
coccus after thorough washing; they are relatively stable, resist- 
ing boiling for prolonged periods, and are present in water or al- 
cohol extracts of muscle, yeast, and vegetable tissue. C represents 
a labile cellular component, inactivated in ten minutes at 65°. By 
itself it is non-reactive with 2 and possesses no reducing power, 
being apparently catalytic in nature. 

Neill 948 observed that both living pneumococci and sterile ex- 
tracts prepared from unwashed cells were capable of oxidizing 
hemoglobin to methemoglobin, and that the action was a reversible 
one, the equilibrium being shifted in either direction by regulation 
of the oxygen tension. Neill 947 also found quantitative evidence of 
the reduction of methemoglobin to hemoglobin by sterile animal 
tissues, such as kidney, testicle, and liver from an exsanguinated 
rabbit. He concluded, therefore, that "the accumulation of large 
amounts of methemoglobin in the circulating blood is probably an 
indication not only of the formation of large amounts of this pig- 
ment, but also of a poisoning of the normal reducing mechanism of 
the animal tissues." This statement is difficult to reconcile with 
that of Stadie (p. 73, ante) that in man, at least, methemoglobin 
is removed from the circulation as rapidly as it is formed. 

In another note, Neill 949 added that in the presence of air the 
oxidizing activity of pneumococci was much stronger than their 
reducing activity in the absence of air, while the reverse was true 
for anaerobic bacteria. From subsequent studies, Neill 950 was able 
to elaborate the graphic representation of the oxidation-reduction 
reaction to include the production of methemoglobin : 



«) 



BIOCHEMICAL FEATURES 81 

+ RH + 2 -» ROOH 



Thermolabile Thermostable Oxidizing agent 

cellular easily oxidized 

substance substance 
or 

C + 2RH + 2 -> 2 R + H 2 2 



6) flOOH (or H 2 2 ) + Hb -* MetHb 

For comparison, Neill developed another formula to represent 
the process of methemoglobin formation during the auto-oxidation 
of other substances: 

(i?H + 2 ->i200H 
fl) (2flH + 2 -»2fl + H 2 2 

b) ROOH (or H 2 2 ) + Hb -> MetHb 

and for the spontaneous formation of methemoglobin in pure 
hemoglobin solutions, in which the iron of the hemoglobin may 
serve as a catalyst, as: 

Hemoglobin -f- x (0 2 ) — » Methemoglobin 

The reactivation of the bacteriolytic activity of oxidized pneu- 
mococcal extracts, according to Neill, Fleming, and Gaspari 958 ap- 
pears as still another effect of the oxidation-reduction phenome- 
non. When oxidized and therefore inactive pneumococcal extracts 
were treated with bacterial reducing agents (anaerobic bacteria), 
the extracts regained their lytic power, and showed a species- 
specificity which argues for their identity with the original bac- 
teriolytic agent. It was held that this agent is an integral part of 
the pneumococcal cell, and that its action is reversible and sus- 
ceptible of being represented by a graphic formula similar to those 
already given. 

Further evidence concerning the reversibility of the action in- 
volved in the transformation of oxyhemoglobin to methemoglobin 



82 BIOLOGY OF PNEUMOCOCCUS 

was supplied by the reports of Schnabel, 1238 and of Schnabel and 
Ninamiya. 1243 Although few new facts were given in these papers, 
the authors found that the activity was not lessened by the addi- 
tion of high concentrations of optochin or sodium glycocholate to 
broth cultures, while, on the other hand, the alkaline reaction and 
reducing power of blood serum and of tissue cells interfered with 
its operation. 

Another effect of oxygen consumption by Pneumococcus is the 
relation between respiration and virulence as described Vy Sevag 
and Maiweg, 1258 who presented the following facts in support of 
their theory: Virulent pneumococci on being transformed into the 
avirulent forms consume much larger amounts of oxygen than do 
the parent organisms, but this gain in activity is only temporary. 
After a time, the avirulent organisms degenerate into forms which 
consume very much less oxygen than do either the virulent or the 
avirulent cells recently derived from the parent strain. 

The phenomenon of oxidation-reduction by Pneumococcus is 
given here in considerable detail because of its important bearing 
on the metabolic process of the cell and because of the thorough- 
ness with which this activity has been studied. It involves the life 
of Pneumococcus ; it affects by inhibition or destruction the lytic 
and synthetic ferments of the bacterial cell ; it is responsible for 
the conversion of the vitally necessary hemoglobin into methemo- 
globin and for the lessened oxygen-carrying capacity of the red 
blood corpuscles ; it may play a part in determining virulence of a 
strain ; and it is undoubtedly concerned in the production of hemo- 
toxin and hemolysin and possibly of other substances harmful to 
the human economy. 

Hemolysin and Hemotoxin 
Pneumococcus, upor lysis, whether natural, or artificially in- 
duced, yields a substance or principle that is actively hemolytic 
for sheep, guinea pig, and human erythrocytes. The substance is 
labile, much of its activity is lost on passing through a filter, and 



BIOCHEMICAL FEATURES 83 

it is destroyed by the action of trypsin. In its properties the he- 
molysin corresponds to the substance contained in the lytic ex- 
tracts of pneumococci that cause the death of guinea pigs on in- 
travenous injection. Its activity is prevented by the presence of 
minute amounts of cholesterol. Following the injection of autoly- 
sates into rabbits and sheep, the serum of the animal acquires in- 
creased power of inhibiting the hemolytic action and therefore Cole 
(1914), 252 whose conclusions these are, believed that the hemoly- 
sin has antigenic properties ; that it is not simply a product of 
autolysis but undoubtedly exists preformed in the pneumococcal 
cell ; it is not given up to the surrounding fluid so long as the 
bodies of the cocci are intact and, accordingly, is to be considered 
as a hemolytic endotoxin. 

Previous as Avell as subsequent to the work of Cole, there have 
been isolated observations of a zone of hemolysis about the colonies 
of pneumococci on blood agar. Libman, 811 in 1905, demonstrated 
an organism, probably a pneumococcus, isolated from the blood of 
a pneumonia patient on the fifth day of the disease, which on blood 
plates produced a peculiar hemolysis. Later (1922) Hewitt and 
Famulener 640 described the production of a zone of hemolysis im- 
mediately around a colony on blood agar of an organism, beyond 
doubt a pneumococcus, isolated from the blood in a fatal case of 
septicemia with meningitis following mastoiditis. From their study, 
Hewitt and Famulener inferred that pneumococci of all serological 
groups, under certain cultural conditions, may hemolyze human 
erythrocytes, and that this property apparently is not influenced 
by the reaction of the medium within the growth limits of the or- 
ganism nor by prolonged refrigeration of the developed colonies on 
blood-agar plates. The authors also expressed the opinion, later 
substantiated by Avery and Neill, that the hemolysin was an intra- 
cellular product liberated from the autolyzed organisms, which dif- 
fuses from the colony into the surrounding blood agar. In contra- 
distinction to the hemolysis of Streptococcus haemolyticus, Hewitt 
and Famulener called the pneumococcal effect "pseudo-hemolysis." 



84 BIOLOGY OF PNEUMOCOCCUS 

Takami 1372 described a zone of hemolysis about the colonies of all 
the sixty strains of pneumococci plated on blood agar. This zone 
was separated from the periphery of the colony by a greenish 
zone ; it appeared on standing at room or ice-box temperature and 
in time might spread over the whole plate. 

Avery and Neill 58 made further studies on this property of 
Pneumococcus. Extracts of pneumococci prepared in broth, or 
from washed pneumococci made with phosphate solution and fil- 
tered through Berkefeld candles, were actively hemolytic for rab- 
bit erythrocytes. The hemolysin was destroyed by ten minutes' 
heating at 55°. The conclusions of the authors were: 

When pneumococcus extracts are exposed to air, the hemotoxin is 
destroyed only in those extracts capable of undergoing auto-oxidation. 
The active agent responsible for this destruction is probably a peroxide 
formed by the union of molecular oxygen with some other easily oxi- 
dizable constituents of the extracts. 

In another study, Neill 051 mixed pneumococcal extracts, in which 
the hemotoxin had previously been inactivated by oxidation, with 
B. coli and anaerobic Bacillus T, sealed and incubated the extracts 
thus treated for several hours. Titration of the bacteria-free su- 
pernatant fluid obtained by centrifuging this mixture showed that 
the inactive oxidation product of pneumococcal hemotoxin was re- 
converted to an actively hemolytic substance by the action of those 
bacteria, which were not themselves hemolytic. The reduction was 
also accomplished by sodium hyposulfite. The active lysin, or hemo- 
toxin, produced by the reduction of the inactive oxidized extracts 
was shown to be identical with the original active hemotoxin, since 
it possessed the same degree of thermolability and was neutralized 
by the same specific antibody. Pneumococcal hemolysin or hemo- 
toxin is, therefore, not an artificial cleavage product but a natural 
substance originating within the cell. Normal serum, egg albumen, 
cholesterol, and peptone, according to Weiss, 1509 inhibit hemolytic 
activity. 



BIOCHEMICAL FEATURES 85 

Returning to the original observation of Cole that active or 
reduced pneumococcal hemotoxin is antigenic, Neill 952 in 1927 re- 
ported that "a neutralizing antibody may be produced by im- 
munization with the hemolytically inactive hemotoxin present in 
oxidized solutions as well as by immunization with the active toxin. 
The antibody is a species-specific antihemotoxin neutralizing the 
hemotoxin from all types of pneumococci." Neill also showed that 
the hemotoxin is independent of the bacteriolytic enzyme present in 
pneumococcal cells. In the next year, Cotoni and Chambrin 282 con- 
firmed the data reported by Cole and by Avery and Neill, and de 
scribed a simple method for the quantitative measurement of the 
hemolytic power of Pneumococcus. While Cotoni and Chambrin 
were able to make satisfactory determinations on a number of 
strains, they reported that the quantity of hemoglobin liberated 
did not follow the law of multiple proportions and that strains 
varied in their hemolytic action. To the authors there appeared to 
be no difference in the susceptibility of the blood cells of man, rab- 
bit, guinea pig, cow, pig, horse, or sheep to pneumococcal hemoly- 
sin. They also gave a method for titrating the antihemolytic 
power of immune serum. 

Sickles and Coffey 1280 published a paper on a hemolytic sub- 
stance in pneumococcal culture broth. No proof was presented 
that this substance is not the same as the hemolysin already dis- 
cussed, but Sickles and Coffey, like Neill, and Cotoni and Cham- 
brin, found that pneumococcal immune serum had a marked inhib- 
itory effect on hemolysis, giving complete inhibition in a 1 to 1,000 
dilution, whereas normal horse serum was effective only in a 1 to 
400 dilution. 

The belief of Cole and his associates that hemolysin is an intra- 
cellular constituent of Pneumococcus was not shared by Cowan. 287 
On the contrary, this author stated positively that the principle is 
extracellular and arises during the growth of the organism in fluid 
media. Its highest titer occurred immediately after the maximal 
period of multiplication — phase of logarithmic growth — while the 



86 BIOLOGY OF PNEUMOCOCCUS 

curves of hemolytic titer and growth seemed to Cowan to denote 
that hemolysin production was not essentially the result of autoly- 
sis. Hemolytic power diminished in old cultures, due to oxidation, 
but could be restored by reducing agents. The hemolysin was not 
type-specific nor was it related to virulence. It filtered readily, was 
rapidly destroyed at 56°, was antigenic, and was produced by all 
but two of twenty-eight strains. The observation that the sub- 
stance appears in broth cultures agrees with that of Sickles and 
Coffey, but it would seem that Cowan had not sufficiently ruled out 
the factors of autolysis to justify the conclusion that the hemoly- 
sin is extracellular in origin. 

While Neill 952 found that pneumo-antihemolysin was without 
effect on the hemotoxins of tetanus and Welch bacilli, Todd 1412 
reported that he was able, to a certain extent, to neutralize pneu- 
mococcal hemolysin and tetanolysin with the serum of horses hy- 
perimmunized against streptococci, the action being ascribable to 
the content of highly active antistreptolysin in the immune se- 
rum. Todd added that the degree of neutralization was not neces- 
sarily correlated with the antistreptolytic titer and that the differ- 
ent hemolysins were distinguishable by quantitative serological 
methods. This partial antigenic overlapping of hemolysins could 
only be demonstrated by the use of hyperimmune serum. 

Purpura Production 
Somewhat analogous to the effects of the hemolysin and hemo- 
toxin produced by Pneumococcus, but probably not a direct con- 
sequence of their action, is the purpuric condition seen in both 
natural and experimental pneumococcal infections. Claude 236 in 
1896 was among the first to note the occurrence of purpura in an 
infant dying of pneumonia. The lesions tended to have black cen- 
tral nodes leading to ulcerative necrosis. But, more important still, 
the causal relation between Pneumococcus and purpura was dem- 
onstrated by the isolation by Claude of pneumococci both from the 
blood and from the center of the purpuric lesions. A similar case 



BIOCHEMICAL FEATURES 87 

was that described by Morse 931 in 1898, in which purpura accom- 
panied a general pneumococcal infection in a twelve-month-old girl. 

A case somewhat similar to Claude's was reported in 1914 by 
Rolland and Buc. 1154 The patient was an infant dying of a pneu- 
mococcal meningitis without lung involvement. Necropsy revealed 
intense visceral purpura, and cultures taken post mortem from the 
meningeal pus and from serous fluid in the purpuric lesions } 7 ielded 
pneumococci identical with a strain isolated from the blood during 
life. Reh 1123 described the case of an infant with bluish-red dis- 
coloration of the right side, left elbow, and the four extremities. 
Bacteriological tests showed that the infection was caused by 
pneumococci. 

Purpura, therefore, is an occasional concomitant of pneumo- 
coccal infections, and the three cases cited are sufficient to illus- 
trate the similarity between this change in the blood with the ac- 
companying capillary permeability and the hemotoxic effects of 
Pneumococcus. The experimental facts are more pertinent to the 
present discussion. 

In 1899, Carnot 199 observed the development of a teinte violacee 
in the nose and ears of a rabbit injected with a pneumococcal 
"toxin." No details of the preparation of the toxin were given but 
Carnot stated that it was sterile. It is probable that he employed 
cell-free broth cultures as did Heyrovsky. 644 The latter, by inject- 
ing culture filtrates into white mice, induced hemorrhagic derma- 
toses and hemorrhages of the mucous membranes and other tissues. 
The condition was severe and affected the internal organs as well 
as the skin. 

Julianelle and Reimann 694 quoted Neill as having observed hem- 
orrhagic purpura in white mice injected with an extract of Pneu- 
mococcus. The authors, by injecting extracts of both virulent and 
avirulent strains into white mice induced a purpuric condition 
manifested after four to six hours as a dark-blue discoloration of 
the skin of the feet, tail, ears, snout, and genitals. Unless the 
amount of extract was large there was no intoxication and the ani- 



88 BIOLOGY OF PNEUMOCOCCUS 

mals recovered. The lesions reached maximal intensity within 
twenty-four to forty-eight hours and disappeared in five to seven 
days. Extracts prepared by the Avery-Neill method were more 
potent than filtrates of pneumococcal cultures. Neither cultures 
younger than eight to sixteen hours nor those that were very old 
were active. Whole, unfiltered cultures rarely caused a reaction, 
while bile-dissolved cultures produced no purpura, although bile 
was shown not to inhibit this activity. The extracts induced pur- 
pura in rabbits and guinea pigs as well as in white mice. Extracts 
of Staphylococcus aureus, Streptococcus viridans and B. coli did 
not exhibit this property. 

The purpura-producing principle withstands heating to 100° 
for ten minutes; it resists oxidation; it is filter-passing; its activ- 
ity is destroyed by digestion with trypsin ; and the substance can 
be obtained from pneumococcal extracts by full saturation with 
ammonium sulfate after the acetic acid-precipitable substances 
have been removed. The principle is common to various pneumo- 
cocci and apparently bears no relation to virulence, nor is it asso- 
ciated with the hemotoxin of Pneumococcus, since the hemolytic 
activity of an extract may be destroyed without affecting the pur- 
purigenic property. 

Julianelle and Reimann concluded that the substance is a deg- 
radation product of Pneumococcus. The authors had in previous 
studies of experimental purpura found that excessive diminution 
of blood platelets, due either to direct destruction of the cells or 
damage to the seat of their origin, was a causative factor. In an- 
other paper, Reimann and Julianelle 1130 reported variations in the 
number of blood platelets in white mice injected with pneumococcal 
extracts. The platelets were greatly diminished after injection, the 
greatest decrease taking place after twenty-four hours. With a 
count of less than 500,000 platelets per cubic millimeter of blood, 
the mice usually developed purpura. A regeneration — an over- 
regeneration — was accomplished by the fourth to the ninth day, 
with a return to normal in about two weeks. The red cells were also 



BIOCHEMICAL FEATURES 89 

greatly reduced in number, but the rate of destruction and regen- 
eration was somewhat slower than that of the platelets, while leu- 
cocytes were slightly, if at all, affected. Reimann and Julianelle 
emphasized the differences between the hemolysin and the purpuri- 
genic principle in pneumococcal extracts. Both the thrombolytic 
and hemolytic properties were destroyed by heating in vitro, al- 
though such heated extracts still were able to produce purpura but 
not a severe anemia in mice. Extracts adsorbed with either blood 
platelets or erythrocytes showed a marked diminution in throm- 
bolytic and hemolytic activity in vitro. Adsorbed extracts, how- 
ever, caused purpura as well as severe anemia and thrombopenia in 
mice. 

In a further study, Julianelle and Reimann 690 offered the follow- 
ing additional conclusions: 

The purpura-producing principle of Pneumococcus is non-antigenic 
in the sense that it does not stimulate the formation of antibodies ; white 
mice acquire an increased resistance to purpura as a result of repeated 
injections of toxic doses of the purpura substance; the serum of rabbits 
immunized with the purified purpura principle, with smooth and rough 
strains of Pneumococcus or with cell extracts, autolysates or the nucleo- 
protein fraction of the organism, does not confer upon white mice pro- 
tection against purpura ; the purpura principle does not exist preformed 
in the cell, but is rather an autolytic derivative, since it is formed only 
when pneumococci undergo autolysis, and it is not found when the auto- 
lytic ferments are inactivated ; the purpura substance is associated with 
the proteose fraction of active pneumococcus extracts. 

Pittman and Falk, 1092 still more recently, substantiated the re- 
sults of Julianelle and Reimann by causing purpura in white mice 
with extracts of pneumococci made by alternate freezing and thaw- 
ing. This extract also protected mice against an infection with 
pneumococci of low virulence when the extract was injected twenty- 
four hours prior to inoculation, but when both extract and culture 
were given simultaneously death ensued. The effect has some resem- 
blances to the action of Bails' aggressin. The strange selective af- 
finity of bacterial proteins for blood cells reminds one of the he- 



90 BIOLOGY OF PNEUMOCOCCUS 

magglutinative action of some vegetable proteins, especially the 
globulins and the proteoses of beans. 

Mair, 856 differing from Julianelle and Reimann, believed that the 
purpura-producing principle was an intracellular substance and 
that the cell must be disintegrated by autolysis or other means be- 
fore the active substance could be absorbed in sufficient amount to 
produce purpura. Another difference reported by Mair was the ac- 
celerating action of the pure bile-salts, sodium desoxycholate or 
sodium choleate, in releasing the purpurigenic principle. By run- 
ning parallel experiments with untreated pneumococcal suspen- 
sions, some bile-treated and others containing sodium desoxycho- 
late, Mair obtained equally good reactions. He inferred from his 
experiments that: 

It may be that solution of the bacterial bodies is all that is required, 
a preformed constituent of the cell thus being set free and rendered 
capable of absorption from the peritoneal cavity of the mouse. On the 
other hand, the delayed reactions obtained with pneumococci which had 
been subject only to slight autolysis, and with pneumococci dissolved 
in bile and immediately heated, suggest the possibility that proteolytic 
changes which may occur in the body of the mouse are required for the 
development of the substance. 

Moreover, Mair found that mice varied in susceptibility to the 
purpura-producing substance or, perhaps, in their ability to effect 
proteolytic changes in the substance. By selecting for breeding 
mice that were sensitive to the purpura reaction, Mair discovered 
that this trait was hereditary, and developed a strain of mice with 
increased susceptibility. 

That the purpura-producing principle might be associated with 
the carbohydrate constituents of Pneumococcus was claimed by 
Wadsworth and Brown 1468 and by Sickles and Shaw. 1283 The injec- 
tion of the cellular carbohydrate intravenously, intraperitoneally, 
or subcutaneously into mice induced a purpuric condition. Not- 
withstanding these observations, there is a reason to believe that 
the effects are ascribable to small amounts of degraded protein in 



BIOCHEMICAL FEATURES 91 

the preparations of cellular carbohydrate. This substance, what- 
ever might have been its nature, in addition to being heat-stable, 
was neutralized by Type I antipneumococcic serum even when the 
latter had been adsorbed with either the homologous soluble spe- 
cific substance or the cellular carbohydrate. Sickles and Shaw re- 
ported that this purpura-producing activity of the polysaccharide 
was destroyed when the carbohydrate was subjected to the action 
of its appropriate carbohydrate-decomposing enzyme, but there is 
no evidence to be found in the communication that the presence of 
proteolytic ferments in the enzymatic extract had been excluded. 
The weight of evidence favors the view that the substance re- 
sponsible for the purpuric manifestations is a cleavage product of 
pneumococcal protein, arising in the lysis of the bacterial cell and 
that purpura production is not a property of any of the compo- 
nents of Pneumococcus as they exist in the normal bacterial cell. 

Virulin, Leucocidin, and Analogous Substances 
Poisoning and dissolution of red blood corpuscles and the elabo- 
ration of purpurigenic substances are not the only ways in which 
Pneumococcus attacks body tissues. Saline extracts of the coccus, 
prepared by Rosenow's method, were reported by Pittman and 
Falk 1092 to decrease phagocytosis of avirulent organisms, to reduce 
the opsonic content of serum, and only slightly to increase the 
virulence of a borderline culture after it had been transferred sev- 
eral times in the presence of the extract. The extracts failed to in- 
fluence the virulence of an avirulent Pneumococcus. The principle 
contained in the extract, prepared by incubating dense suspen- 
sions of pneumococci in small volumes of isotonic sodium chloride 
solution with subsequent heating and centrifuging, the authors 
named "Virulin." Considering the feeble action of the substance in 
raising the virulence of pneumococci, the name would seem to be 
too specific a designation for an unidentified substance. 

Another toxic principle of Pneumococcus is the "Leucocidin" 
described by Oram, 1032 who reported that in actively growing cul- 



92 BIOLOGY OF PNEUMOCOCCUS 

tures of Pneumococcus a toxin was produced that, as demonstrated 
by the Neisser and Wechsberg method, destroyed leucocytes. Leu- 
cocidin was present in aerobic and anaerobic cultures of both viru- 
lent and avirulent strains of Types I, II, and III. It was easily 
oxidized, but was not injured by exposure to 70° for one hour. The 
toxin in the preparation was not destroyed by evaporation in a 
vacuum at 5°, and its action could be enhanced three to four 
times by the addition to the culture of laked red blood cells. Ap- 
parently the same cultural conditions governed the production and 
preservation of both the hemotoxin and leucocidin, although the 
latter was distinguishable by its greater thermostability and its 
appearance in cultures where no hemotoxin could be demonstrated. 
The filtrates containing active leucocidin were not toxic for mice in 
two cubic centimeter amounts injected intraperitoneally, and re- 
peated doses of the filtrate gave only slight protection. Simultane- 
ous injections of the extract and avirulent pneumococci did not 
raise the virulence of the strain, so that the leucocidin does not 
share this alleged action of the so-called virulin. Sterile superna- 
tant fluids of phenolized exudates of rabbits with empyema, while 
in themselves not toxic, upon repeated intravenous injection pro- 
duced an antileucocidin. Normal rabbit serum did not neutralize 
the pneumococcal leucocidin, whereas the majority of human se- 
rums tested possessed neutralizing properties. 

One more morbid effect elicited by pneumococcal extracts or 
autolysates deserves attention and that is the necrotizing principle 
reported by Parker. 1059 " 60 This principle is a filtrable substance, is 
extremely thermolabile, is sensitive to oxidation, and can be sepa- 
rated from hemotoxin by adsorption with red blood cells, since the 
necrosis-producing principle remains unaffected after removal of 
the hemotoxin. The necrotizing substance or substances obtained 
from both Types I and II pneumococci are neutralized by Type I 
antipneumococcic serum, and hence the necrotizing substance is 
not type-specific. 

The virulin, leucocidin, and, to coin a name, the necrotin have 



BIOCHEMICAL FEATURES 93 

been described in some detail as representing one of the recent 
trends in the investigation of Pneumococcus and to draw attention 
to still unstudied possibilities of this remarkable cell and its prop- 
erties. 

So-Called Toxins 

The severe intoxication that so frequently accompanies pneumo- 
coccal disease naturally led early investigators to search for a 
soluble toxin. Profoundly impressed, as they must have been, by 
the discoveries by von Behring and Kitasato and by Roux of 
diphtheria and tetanus toxins and of the dramatic efficacy of the 
corresponding antitoxins, there came a vision of a new and similar 
agent for the treatment of pneumonia. 

Foa, 458 in 1890, thought that he had found a poison in pneumo- 
coccal cultures. He isolated the supposed principle by precipita- 
tion with ammonium sulfate, dialysis, and subsequent concentra- 
tion. The extract thus prepared produced marked biological 
changes in rabbits, but failed to kill the animals. Repeated injec- 
tions raised the resistance of the test animals to later inoculation 
with a virulent culture, but it seems safe to infer that Foa was 
merely dealing with the deleterious degradation or lytic products 
of Pneumococcus. In the next year, Bonome 137 succeeded in pre- 
paring filtrates that were lethal for rabbits. The toxicity was 
directly related to the virulence of the culture, and the more poi- 
sonous extracts, after repeated doses given subcutaneously, intra- 
venously, and intraperitoneally, rendered rabbits resistant to in- 
fection. But the subsequent effects were neither toxic nor antitoxic 
in the true sense of the terms. Issaeff, 673 at the Pasteur Institute, 
reported similar experiments with comparable results. The serum 
of rabbits treated with broth cultures of low toxicity possessed 
therapeutic but no antitoxic power. Issaeff, therefore, had not dem- 
onstrated the existence of a toxin. Bunzl-Federn 186 also tested in a 
similar way heated broth cultures but found that their toxicity 
and antigenicity were feeble. Cole 250 was able to elicit more severe 



94 BIOLOGY OF PNEUMOCOCCUS 

and fatal toxic or poisonous effects with saline extracts and bile 
solutions of pneumococci. The intravenous injection of the latter 
materials into guinea pigs initiated a train of symptoms terminat- 
ing in a type of death resembling that seen in acute anaphylaxis. 

In the same year, Rosenow 1166 published a series of four papers, 
in which he described observations similar to those of Cole, accom- 
panied by an explanation of the observed phenomena. In the first 
article Rosenow reported that a single injection of saline autoly- 
sates of Pneumococcus, Pneumococcus-leucocyte mixtures, pneu- 
mococcal exudates, and mixtures of the organism with normal or 
immune serum, all produced identical symptoms in normal guinea 
pigs, and that the symptoms were those of acute anaphylaxis. The 
serum of animals sensitized to Pneumococcus when allowed to act 
on the organisms produced this toxic substance more rapidly than 
did normal serum, and this toxin production as measured by the 
polariscope was accompanied by a more rapid proteolysis. Mor- 
phine, ether, urethan, atropin, and adrenalin protected normal 
guinea pigs against the poisonous action of the preparations just 
as they protected sensitive pigs on re-injection. Rosenow's conclu- 
sions are significant: 

The behavior of normal and sensitized guinea pigs toward unauto- 
lyzed extracts of pneumococci, which are non-toxic to the former and 
very toxic to the latter, toward partially autolyzed extracts which are 
very toxic to the former, and slightly or not at all to the latter, and 
toward more completely autolyzed extracts, which are non-toxic to both, 
speaks strongly in favor of the view of a rapid parenteral digestion 
into toxic cleavage products in sensitized animals. 

He added that the cleavage products formed in vivo were identical 
with the toxic substances obtained in vitro, and that the appear- 
ance and disappearance of toxicity seemed to be definitely related 
to proteolysis. Rosenow also reported that a single intravenous 
injection of non-fatal doses of extracts before they had become 
toxic, or while highly toxic, and especially after the toxic stage 
had been passed, or of autolyzed pneumococci, rendered guinea 



BIOCHEMICAL FEATURES 95 

pigs insusceptible to subsequent injections of toxic pneumococcal 
autolysates. Although the duration of this protection was not men- 
tioned, the statements point to a refractory state or temporary 
tolerance so characteristic of degraded proteins and not to an im- 
mune condition. 

In a second communication, Rosenow 1167 described other charac- 
ters and properties of the poisonous substance. It was destroyed 
when the clear autolysate was heated for twenty minutes at 60°, 
as well as by weak solutions of hydrochloric acid ; it was adsorbed 
by animal charcoal from which it could be recovered by ether. 
From its chemical behavior the author decided that the toxin was 
probably a base containing amino groups. He concluded by say- 
ing, "Indications have been obtained showing that during pneumo- 
coccus infections toxic substances are produced which do not call 
forth any immunizing response." 

In a third paper, Rosenow 1168 ascribed the production of the 
toxic material to the action of a proteolytic enzyme present in ex- 
tracts and autolysates of the pneumococcal cell, and in a conclud- 
ing report 1169 described the action of various pneumococcal prod- 
ucts on dogs. The symptoms and lesions were strikingly like those 
observed in immediate canine anaphylactic shock. The hemor- 
rhages, especially those in the intestines, the effect on the respira- 
tion, the extreme degree of cyanosis, the delayed coagulation of the 
blood, and the presence of carbon dioxide in the stomach indi- 
cated to Rosenow that the chief effects of the toxic substances 
were due to an interference with normal oxidative processes. The 
action of the autolysates closely resembled that of protein cleav- 
age products and, according to Rosenow's view, it made no essen- 
tial difference whether the poisonous substances were formed in 
vitro, in the consolidated lung in man, or at once in sensitized 
dogs, since they were all of the same general nature and their ef- 
fects differed only in degree and not in kind. 

The anaphylactoid shock seen after the intravenous injection of 
pneumococcal extracts into guinea pigs has also been reported by 



96 BIOLOGY OF PNEUMOCOCCUS 

Weiss, 1509 who obtained an analogous reaction in rabbits. He saw 
no parallelism between the toxicity and hemolytic power of the 
extracts, nor any neutralizing action by antipneumococcic se- 
rum. Weiss, like Rosenow, found a similar toxic principle in lung 
exudates of patients suffering from acute lobar pneumonia. 
Clough's 241 paper (1915) contained similar observations. In the 
same year, Boehncke and Mouriz-Riesgo 134 failed to isolate the 
toxic substance from young cultures or from sodium taurocholate 
solutions of virulent strains. Contrary to earlier reports, these two 
authors found that the serum of rabbits treated with these prepa- 
rations displayed antitoxic action but did not affect the infective 
process. There followed a paper by Weiss 1509 on "Pneumotoxin," 
in which he repeated Cole's observations on the anaphylactoid re- 
action caused by sodium choleate solutions of living, virulent, 
washed pneumococci. The description of the properties of the 
preparations agrees with previous accounts of the characters of 
pneumococcal hemotoxin. 

Chesney and Hodges 221 departed from the idea that the intoxi- 
cation in pneumonia was due to a toxin, believing that it was more 
reasonable to associate the phenomenon with the growth of the 
organisms, rather than with their death and dissolution. The au- 
thors' attempts to detect toxic substances in fluid cultures of pneu- 
mococci during the period of active growth proved entirely nega- 
tive. 

The demonstration of a toxin was claimed by Olson 1029 who ex- 
perimented with sterile sodium ricinoleate solutions of pneumo- 
cocci. He also claimed that the intraperitoneal injection of such 
preparations produced cutaneous reactions in animals and caused 
clinical and pathological symptoms of pneumonia. An antiserum 
against this substance appeared to prevent to a high degree both 
pulmonary and cutaneous reactions. Olson stated without qualifi- 
cations that a toxin had been demonstrated, that the action of the 
immune serum was antitoxic, and that "indications are that the 
toxin may be of value in the production of active immunity to 



BIOCHEMICAL FEATURES 97 

pneumococcus infections." In the light of later events this claim 
and hope would seem to be premature. Larson, 787 with pneumococ- 
cal filtrates supplied by Olson, found the same congestion of the 
lungs in white mice injected intraperitoneally. Of skin tests per- 
formed with the filtrates, those on pneumonia convalescents were of 
possible significance in being uniformly negative. Larson voiced the 
same conviction that the filtrates contained a soluble toxin. 

In a second paper, Olson, 1030 after describing the lesions pro- 
duced by pneumococcal filtrates in mice, went so far as to say that 
the toxin was an exotoxin and not an autolytic product or endo- 
toxin, and gave as his reason that eight-hour culture filtrates pos- 
sessed marked toxicity but contained little hemotoxin. The toxic 
principle was relatively thermostable, but was completely destroyed 
by boiling for one hour. While unpreserved lots lost activity, 0.3 
per cent cresol served to maintain the toxicity for several months 
regardless of the temperature. Active immunization of mice by se- 
rial injections of the toxin resulted in protection against 1,000 to 
10,000 fatal doses of pneumococci, while the specificity of the im- 
munity induced bore no relation to the serological type of the or- 
ganism from which the so-called toxin was obtained. The stability 
of the toxic principle in the presence of heat, the stabilizing action 
of cresol, and the lack of specificity are, as we know, characters 
not shared by true exotoxins. 

The report of Parker and Pappenheimer 1063 takes us back to the 
toxic action of autolysates. Solutions were prepared by allowing 
a suspension of pneumococci, sealed in tubes under vaseline, to 
stand in the dark at 22° to 24° for two to five days and then in the 
ice-box. The autolysates were centrifuged in the cold, the seals 
opened, and the clear supernatant fluid passed through a well-iced 
Berkefeld filter. The filtrate injected intratracheally in a dose of 
0.2 cubic centimeters was highly toxic for guinea pigs, death tak- 
ing place either within a few hours or within three days. In animals 
succumbing early, there was intense hemorrhagic edema of the 
lungs with beginning inflammatory reaction ; in animals surviving 



98 BIOLOGY OF PNEUMOCOCCUS 

for eighteen hours or longer, extensive areas of pneumonia were 
found. As a control, the authors made intratracheal injections of 
living, virulent pneumococci which were followed by a transient, 
slight lesion and recovery, or later by death from septicemia with- 
out pneumonic lesions. The addition, however, of a sublethal dose 
of toxic autolysate to the living pneumococci altered the reaction 
of the animal so that an extensive pneumonia developed associated 
with unrestrained multiplication of the organism. This synergistic 
property was far more pronounced than that of the virulin of 
Pittman and Falk and was more like that of an aggressin. 

Yamamoto (1929) 1557 listed unheated pneumococcal filtrate, 
heated filtrate, and "standard pneumococcus vaccine" (?) accord- 
ing to toxicity in a ratio of 1 to 2 to 6, but none of the three was 
notably toxic, not less than 6.0 cubic centimeters of the last-named 
preparation being required to kill a rabbit. 

In 1929, Parker 1060 returned to a study of the necrotizing sub- 
stance of Pneumococcus in comparison with the lung-toxic princi- 
ples in autolysates. She found both substances to be sensitive to 
heat and to oxidation, and both were neutralized by the same anti- 
autolysate serums. The lung-toxic principle, however, was ab- 
sorbed or inactivated by red blood cells, whereas the necrotizing 
principle was not. In the discussion, Parker wrote: 

Since pneumococcus hemotoxin is present in the anaerobic autolysate 
and is also absorbed by red cells, it seemed possible that it was this 
substance in the autolysates which caused the diffuse lung lesions and 
death of guinea pigs. However, it was found that the intratracheal in- 
jection of pneumococcus hemotoxin prepared by the method of Avery 
and Neill only occasionally produced the characteristic reaction caused 
by the intratracheal injection of the anaerobic autolysates. From these 
experiments we believe, therefore, that the necrotizing and lung-toxic 
principles, and probably the pneumococcus hemotoxin also, are all sepa- 
rate entities in the anaerobically produced autolysates described. 

Pittman and Southwick 1093 were also attracted by this problem. 
In their experiments, the injection into mice of extracts produced 



BIOCHEMICAL FEATURES 99 

by repeatedly freezing and thawing pneumococci was followed by 
marked hemorrhagic lesions on all or a part of the external sur- 
face that was free from hair, and at necropsy hemorrhagic areas 
could be found in practically every tissue of the mouse. The adju- 
vant action of the extract was seen when mice, dying after injec- 
tion with extract followed several hours later by culture inocula- 
tion, developed more marked hemorrhagic lesions than any other 
group of mice studied. Fresh filtrates of virulent pneumococci pro- 
duced slight pathological reactions in mice but, when the filtrates 
were injected with a culture of low virulence, 71 per cent of the 
mice at necropsy showed a fibrino-purulent pleuritis. The results 
might be interpreted as pointing to the production of an aggres- 
sin-like substance in the early stages of pneumococcal growth with 
the development of a more violent poison as growth continues and 
the cells undergo autolysis. 

Jamieson and Powell 676 also described a skin-reacting substance 
in pneumococci. To them its properties were comparable to those 
of the toxins of some streptococci. The alleged success in develop- 
ing a neutralizing antiserum led the authors to believe that the 
present type of antibacterial immune serum would be more valu- 
able if it contained these antiskin-toxic elements. Sabin (1931), 1203 
investigating the part anaerobic autolysates of Pneumococcus 
play in the course of natural infection, came to conclusions dia- 
metrically opposed to those of Parker and the other students shar- 
ing her ideas. Sabin wrote: "The only conclusion that may be 
drawn, however, is that the anaerobically produced toxins prob- 
ably do not play any part in the causation of death of mice in- 
fected with very large doses of pneumococci." The failure of "anti- 
pneumotoxic" serum to modify the course of pneumococcal infec- 
tion led Sabin to conclude that: "It seems fair to assume that the 
anaerobically produced toxins are probably products primarily of 
the enzymatic changes occurring in in vitro autolysis, and play no 
part in natural infections." The idea of producing an immune se- 
rum capable of neutralizing the poisonous principles of Pneumo- 

/ 



100 BIOLOGY OF PNEUMOCOCCUS 

coccus still persists. Coca, 246 after continuing the investigation of 
the toxic principle in Pneumococcus, announced additional data in 
1936. When the pneumococcal cultures were grown in the presence 
of oxygen a toxic substance was formed but it was devoid of anti- 
genic properties. When, however, carbon dioxide was supplied to 
the growing cultures, the sterile filtrates were not only toxic but 
antigenic. According to Coca, the pyrogenic toxin seemed to be 
type-specific and was not the same as the type-specific polysac- 
charide. Evidence was presented purporting to show that the in- 
jection of the toxic filtrates into the human body or pneumococcal 
disease in human subjects provoked neutralizing substances in the 
serum for filtrates of the homologous type, which the author be- 
lieved were specific antitoxins. 

A careful reading of original papers dealing with the subject of 
toxin production by Pneumococcus fails to bring conviction that 
the substances or extracts described should be looked upon as 
true soluble toxins. The principle involved in their formation or 
preparation, that is, disintegration of the pneumococcal cell by 
self-proteolysis, bile solution, or freezing and thawing; the nature 
of the local and systemic reactions the materials evoke; the acute 
anaphylactoid death produced in guinea pigs ; and the feeble anti- 
genic powers, all argue for the classification of the so-called toxins 
with the proteins of smaller molecular size formed in the hydrolysis 
of whole proteins. One has only to recall the work of Vaughan 1447 
and of others on the effects of the cleavage products of bacterial 
and of pure proteins, and the many studies on proteoses and pep- 
tones, to be impressed by the similarity between the alleged pneu- 
mococcal toxins arising from the autodigestion of the somatic pro- 
tein of Pneumococcus and protein poisons. The slight antigenic 
power of the pneumococcal derivatives and their seeming lack of 
specificity is easily explained when one remembers that the hy- 
drolysis of proteins is not always an orderly, step-by-step process. 
Along with the formation of proteoses and peptones, or polypep- 
tids and amino acids there may be traces of proteins of larger 



BIOCHEMICAL FEATURES 101 

molecular size, even to whole unsplit proteins, remaining in diges- 
tion mixtures. Traces of intact proteins acting as antigens may be 
responsible for the weak neutralizing power of the serum of ani- 
mals treated with extracts, filtrates, and autolysates of Pneumo- 
coccus or, what is more probable, such feeble and transient resist- 
ance as these antigens induce is merely the tolerance established by 
degraded proteins and not immunity. 

While it is possible that a true toxin may be a constituent of the 
pneumococcal cell or a product of its metabolic processes, until 
more convincing evidence is presented, it would seem reasonable to 
look upon these so-called "toxic" effects as being referable to the 
action of bacterial protein poisons. 

Summary 

It may assist the reader to gain a clearer conception of the rela- 
tion of the biochemical phenomena manifested by this bacterial 
cell to the morbid processes induced by the living organism to se- 
lect from the mass of data the more significant discoveries and to 
weave them together into a summary. Pneumococcus is a fragile 
body and contains within itself enzymatic forces that lead to its 
disruption and disintegration, rob the substrate in which it lives of 
nutrient substances, and from these substances evolve chemical 
agents that arrest further growth, cause the death of the organ- 
ism, and affect the cells of the animal body into which the microbe 
may find its way. 

Intracellular proteases break down the proteins of the cell and 
of the substrate upon which the cell feeds into smaller fragments, 
which in turn are still further reduced in size by the same ferment 
or possibly by a peptidase. This proteolysis reduces the food sup- 
ply in the medium, and the products of the digestive action may in- 
duce purpura and cause other more or less violent toxic effects in 
the animal host. The poisonous principles, as far as can be learned, 
are not true toxins, but resemble degraded proteins in their physio- 
logical action. Pneumococcus is also endowed with saccharolytic 



102 BIOLOGY OF PNEUMOCOCCUS 

enzymes, capable of attacking starch, inulin, and glycogen ; invert- 
ases that convert complex saccharides into simpler sugars, and 
other ferments that split these sugars into acids, while the acid so 
formed arrests further proliferation of the bacterial cell. Intra- 
cellular lipids are converted into fatty acids by pneumococcal 
lipases and therefore the self-destruction of the cell may be com- 
plete. Furthermore, the ingredients of the medium in which the or- 
ganisms grow are largely replaced by a new order of constituents. 
The action of all these enzymes may be reversible and by their ac- 
tion or that of some similar agents Pneumococcus is able to build 
protein, lipids, and somatic and capsular polysaccharides from 
substances present in the substrate. 

In the operation of these vital processes, oxygen plays an im- 
portant part. The element is essential to the hydrolysis and syn- 
thesis of protein, lipid, and sugar but it may act in a variety of 
ways. Some of the products of oxidation are inimical to the nor- 
mal functioning of the bacterial cell and some affect changes in the 
respiratory mechanism of the blood. Pneumococcus utilizes oxy- 
gen to form peroxide that is toxic to the cell. Peroxide destroys the 
labile constituents of the pneumococcal cell, which with the easily 
oxidizable intracellular substance continue to form an oxidizing 
substance responsible for the conversion of oxyhemoglobin into 
methemoglobin. 

Pneumococcus, therefore, by the action of its intrinsic enzymes 
may destroy itself and by the operation of its oxidation-reduction 
system and its extracellular derivatives or products may, through 
the dissolution of red cells, the conversion of oxyhemoglobin into 
methemoglobin, and the lowering of oxygen capacity of the blood, 
wreak serious damage to the tissues and pervert the normal physio- 
logical functions of the body of the infected host. 



CHAPTER IV 
CLASSIFICATION OF PNEUMOCOCCI 

The separation of the members of the species into specific types 
by immunological reactions, with a description of methods for the 
determination of these serologically specific types. 

BY tinctorial and cultural methods it is a relatively simple task 
to differentiate pneumococci from other bacterial species but 
it is impossible by these methods, except in the case of Type III 
organisms, to distinguish pneumococci of one serological type 
from those of another. 

The lance-shaped form, with the presence or development of a 
capsule, and the appearance of the colonies on blood agar, with 
their zone of greenish discoloration, are presumptive signs of the 
identity of these cocci. Solubility in bile, fermentation of inulin, 
marked pathogenicity for mice and rabbits, and sensitiveness to 
optochin are all characters which make possible a positive identifi- 
cation of the species. The possession of these characters has defi- 
nitely placed the former bile-soluble Streptococcus mucosus among 
the pneumococci, and its mucoid growth, thick capsule, and greater 
virulence serve to separate it from pneumococci of some of the 
other types. However, it is by their serum reactions that the sepa- 
ration of pneumococci into definite immunological types has been 
accomplished. 

Serological Classification: 1898-1932 

It was undoubtedly Bezancon and Griffon 108 " 10 who, in 1897, 
were the first to report a discovery that later was to establish dif- 
ferences in the serological behavior of pneumococci as a basis for 
an invaluable aid in all phases of research on Pneumococcus. While 
Metchnikoff 894 and later Mosny 933 had previously observed the ag- 
glutinative action of antipneumococcic serum, it was Bezancon and 



104 BIOLOGY OF PNEUMOCOCCUS 

Griffon who announced that from the standpoint of agglutination 
there existed several races of pneumococci that behaved serologi- 
cally as though they were different microbes. 

At about the same time, Mennes (1897), 893 with a highly potent 
serum prepared by the immunization of goats and horses with a 
single strain of Pneumococcus, was able to demonstrate wide 
agglutinative coverage for other strains irrespective of their origin, 
whether from cases of pneumonia or from normal mouths. Kind- 
borg* took the view that pneumococcal antibodies, whether agglu- 
tinins or protective antibodies, were strictly strain-specific. Two 
years later, Eyre and Washbourn 376 confirmed Bezancon and Grif- 
fon's discovery by means of another immunological reaction. When 
they tested the protective action of a specific immune serum 
against five strains of pneumococci, they obtained excellent pro- 
tection in mice against four of the strains, but none against the 
fifth. From the experience, the authors concluded, "There exist va- 
rieties of pneumococci which at present are only distinguished by 
the action of antipneumococcic serum." 

In the next year, Bezancon and Griffon, 113 continuing their 
study of the agglutination reaction, noted that the serum of a 
pneumonia patient was most active against the particular strain 
causing the infection. Among the experimental data was included 
the description of a strain that must have been a Type III pneu- 
mococcus, since its unusually large capsule and the white, viscous 
peritoneal exudate it caused in white mice were characteristic of 
that type. The authors said that the strain was also distinctive in 
its serum reactions, but unfortunately did not specify in what re- 
spect. In a separate paper a year later (1901), Eyre 371 reported 
that agglutinability of pneumococci depended upon an optimal re- 
action of the medium in which the organisms were grown. This ob- 
servation was partly confirmed by Yoshioka 1564 in 1923. 

The contribution of Collins (1905) 270 to the serological differ- 
entiation of pneumococci seems to have been overlooked by some 

* Quoted by Neufeld and Schnitzer. 



CLASSIFICATION OF PNEUMOCOCCI 105 

authors and reviewers. She prepared agglutinative serum by im- 
munizing rabbits with repeated doses, first of killed, then of living, 
and finally of killed broth cultures of pneumococci. The strain used 
as an antigen must have belonged to a type other than I or II, 
since the serum agglutinated only a few of the seventy strains 
tested. Collins' results may be summarized as follows: Pneumo- 
cocci by reason of their agglutinating properties exhibit a tend- 
ency to separate into numerous groups ; Pneumococcus mucosus 
forms a distinct and consistent race, and the resistance of the ag- 
glutinins produced by it to absorption by streptococci indicates a 
nearer relation to Pneumococcus than to Streptococcus. There 
was considerable uniformity of reaction of the various strains in 
low dilutions, but this uniformity was not continued as the animal 
became more highly immunized, and it was not possible for Collins 
to establish a definite relationship, between the agglutination reac- 
tion and the other characters of Pneumococcus except in the case 
of Pneumococcus mucosus. 

In 1906, Eyre, Leathern, and Washbourn 372 suggested the sepa- 
ration of pneumococci into two groups according to the reaction 
of rabbit tissue to infection, that is, a fibrinous type and a cellu- 
lar type, but this idea apparently was never pursued. 

It remained for Neufeld and Haendel 991 in 1910 to demonstrate 
the full significance and the differential value of these phenomena. 
With a collection of pneumococcal strains isolated from a series of 
pneumonia patients the authors obtained, by the immunization of 
rabbits, asses, and horses, monovalent serums of high potency. 
With a culture isolated in 1909 a serum was prepared that pro- 
tected mice against the majority of the other strains, and this 
strain was called "Pneumococcus I." All the pneumococci against 
which the serum protected mice were classed as "typical," and all 
others as "atypical." Among the latter was one strain, "Franz," 
that was later found to be Type II. The "typical" antiserum had 
no effect on the "Franz" strain, and a "Franz" antiserum failed 
to protect mice against the Type I culture. Neufeld and Haendel 



106 BIOLOGY OF PNEUMOCOCCUS 

found that the results obtained by protection experiments agreed 
with those of the agglutination reaction. On the basis of these dis- 
coveries Neufeld and his colleague were the first to recommend that 
serums should be developed for all types, since it was thought that 
there were probably many types and that a given serum always 
exhibited type-specificity. For the serum treatment of pneumonia 
the authors suggested that the agglutination test be employed to 
determine the type of the infecting organism before administering 
the specific serum. 

The next impetus to serological classification of pneumococci 
came in 1913 from the work of Dochez and Gillespie. 322 These au- 
thors, by the methods of protection and agglutination, divided the 
species into four groups. Groups I and II included over 60 per 
cent of the strains tested, Group III consisted of organisms of the 
Pneumococcus mucosus type, and Group IV was a heterogeneous 
collection of strains that fell into none of the first three divisions 
and that reacted only with strictly homologous, that is, individual, 
strain-specific serum. When fourteen different cultures were tested 
against eleven serums for these heterologous strains, no cross-pro- 
tection was seen, except in one instance.* 

Dochez and Gillespie's Type I Pneumococcus corresponded to 
the strain originally isolated by Neufeld and Haendel in 1909 and 
sent by the latter authors to the Rockefeller Institute. The Ameri- 
can Type II was identical with Neufeld and Haendel's "Franz" 
culture, which was a representative of the most commonly occur- 
ring of the atypical strains. Group IV of the American authors 
Neufeld preferred to call Group X, and this designation is often 
found in the German literature. 

The typical and atypical strains of Neufeld and Haendel had, 
therefore, now been sorted into three definite and specific types and 
a heterogeneous group of wide immunological diversity. 

* Wirth,i524 in a study (1927) of pneumococci from infections of the ears, 
sinuses, and throat, found strains of the mucosus type which, irrespective of 
the presence or absence of slimy growth on blood agar, agreed in their aggluti- 
native characteristics with those of the American Type III. Wirth recom- 
mended that the name Streptococcus mucosus therefore be abandoned. 



CLASSIFICATION OF PNEUMOCOCCI 107 

Lister (1913), 815 studying pneumococci recovered from native 
laborers in South Africa, was struck by the comparative rarity 
with which recently isolated pneumococci were opsonized even by 
serum of pneumonia patients. He succeeded however in obtaining 
opsonic preparations which showed massive agglutination and 
marked phagocytosis. Tests on his first four cases suggested the 
"existence of groups of pneumococci, having distinct serological 
reactions," and the "presence of specific agglutinins in 'critical' 
sera from pneumonic patients." Serum from a Group I case failed 
to react with cultures of Group II, III, or IV pneumococci, and 
serum for Groups II, III, and IV failed to react with a Group I 
culture, but any of the Group II, III, and IV serums reacted typi- 
cally with any of the cultures of Groups II, III, and IV. The nu- 
merals are Lister's and bear no relation to the designations used 
by Dochez and Gillespie. It was not until the completion of his 
work that Lister learnt of the earlier classification. He immedi- 
ately compared his groups with those of the American authors, 
and divided his twenty cultures into five groups, A, B, C, D, and E, 
of which Group A could not be identified with any of the American 
types, while Group C corresponded to Type I, and Group B to 
Type II. Lister's E group was later found to be the same as Type 
III. 

A year later, Lister 816 was able to clarify the apparent confu- 
sion existing between his classification and that of Dochez and 
Gillespie. He sent to the American workers dried spleens of mice 
infected with cultures representing his groups and received from 
them in return samples of Type I, II, and III serums. This mutual 
investigation showed that Lister's C and B groups corresponded 
respectively with the American Types I and II. Since Lister had 
no E cultures at the time, he could not correlate it with any of the 
American groups. At the Hospital of the Rockefeller Institute, 
however, the identification of the E strain as Pneumococcus III 
was made. In regard to Dochez and Gillespie's Group IV, Lister 
stated that it was at this point that his classification departed 



108 BIOLOGY OF PNEUMOCOCCUS 

widely from that of the workers at the Rockefeller Institute. In the 
Transvaal there were a number of additional groups, and Lister's 
A was of great importance, since its members were more prevalent 
than those of any of the groups C, B, or E, and caused a higher 
case fatality. His unclassifiable groups D, F, G, and X Lister con- 
sidered of less importance, because of the fluctuation in their 
prevalence and low case mortality. The pneumococci in these 
groups showed strain-specificity with no cross-reactions in agglu- 
tination. Lister's A and D Group cultures were placed by Dochez 
and Gillespie in Group IV. 

Dochez and Avery, 319 in their 1915 paper, explained that pneu- 
mococci of Groups I, II, and III were found principally in asso- 
ciation with disease and were distinctly parasitic in type, while 
Group IV comprised a heterogeneous series of strains, not related 
antigenically, which caused a minority of cases of pneumonia, and 
from which the pneumococci occurring in the normal mouth were 
indistinguishable. Then Avery, 33 by means of agglutination and 
protection tests, further divided Type (originally Group) II 
pneumococci into three subgroups, designated by him as "HA," 
"LIB," and "IIX." Protection tests showed that organisms of any 
subgroup were not protected against by the serum of other sub- 
groups, nor did the strains absorb from Type II antipneumococcic 
serum the specific immune bodies of the other subgroups. Aver}' 
stated that the organisms of the three subgroups were biologically 
related to Type II Pneumococcus, that organisms of subgroups 
HA and IIB were characterized by immunity reactions identical 
within the respective subgroup, but that subgroup IIX consisted 
of heterogeneous strains which did not cross with other strains or 
with Types IIA or IIB. 

In 1916, Olmstead 1027 after testing the agglutinative reaction of 
over two hundred strains of pneumococci isolated from normal and 
infected human beings against fifteen serums, including Type I and 
Type II serums, against which all those strains failed to react, 
confirmed the validity of the classification of pneumococci into 



CLASSIFICATION OF PNEUMOCOCCI 109 

Types I, II, and III and then divided the members of Group IV 
into at least twelve groups, some of which contained subgroups. In 
a subsequent report, Olmstead 1028 suggested that some members of 
these groups served as connecting links between Type II and 
Group IV, and, because of a closer relationship with the latter, 
should be classed as Pneumococcus IV rather than as IIX. The 
same proposal was made by Clough, 238 who after identifying 
strains of Avery's subgroups IIA and IIB among 121 cultures 
isolated from cases of lobar pneumonia, suggested that, since mem- 
bers of these two subgroups possessed relatively low virulence for 
animals and had been recovered from the mouths of normal per- 
sons, the strains would be found epidemiologically to resemble 
Group IV organisms more closely than those of Types I or II. 

Nicolle, Jouan, and Debains 1011 did not accept Group IV and the 
American classification, claiming that a large number of pneumo- 
cocci studied by them were not agglutinated by their own or 
American serums, except when treated with dilute hydrochloric 
acid according to the method of Porges. The authors recommended 
that the conception of Type IV — a purely negative type — ought 
then to be abandoned henceforth. 

Nicolle with Debains 1009 again studied by the agglutination re- 
action a large number of pneumococci from varied sources. They 
used the American classification and their results showed that the 
strains, as studied, varied in agglutinability from a complete ab- 
sence of this property to spontaneous clumping in normal horse 
serum. 

In a review of various immunological reactions, Nicolle and De- 
bains (1920) 1010 concluded that races of Pneumococcus — "anti- 
genic races" — could not be determined by agglutination alone. 
Their work is, in one respect, reminiscent of Gillespie's 516 data on 
the acid agglutination of pneumococci. Tested by this method, 
strains belonging to Types I and II had, as a rule, narrow zones 
of agglutination. Other pneumococci had broad zones, or in a few 
cases, narrow zones not coincident with those occupied by organ- 



110 BIOLOGY OF PNEUMOCOCCUS 

isms of the fixed types. The acid-agglutination of the majority of 
pneumococci of Types I and II was extremely susceptible to the 
inhibiting action of salts, but this was not true of other pneumo- 
cocci. These influences, with the variations they cause, while of a 
certain philosophic interest, have little bearing on the practical 
application of agglutination to the classification of members of 
this bacterial species. 

Following the lead developed by Avery, Stillman 1327 further sub- 
divided Type II strains into twelve distinct groups. These were 
designated as Ila to Ilm. Groups Ilb-c-f-m originated in human 
mouths. Groups Ila and Ilh were encountered largely in connec- 
tion with disease, while the general fatality of acute lobar pneu- 
monia due to these atypical Type II pneumococci was fairly high. 
Stillman's tables, showing the percentage of incidence and of 
mortality of strains of these subgroups, offer an interesting com- 
parison with those given later by Cooper and her associates. They 
serve to emphasize the close relation which exists between some 
members of the Type II and those of the new types formerly in- 
cluded in Group IV. 

In conformity with Stillman's and Olmstead's investigations, 
Christensen proved that Type IVm of Stillman also constituted a 
heterogeneous group which could be further divided into subordi- 
nate groups. Christensen 228 " 9 carried out comparative tests by 
means of agglutination, agglutinin-absorption, and complement 
fixation and gave preference to the simple agglutination technique 
as being the most convenient and efficient method for serological 
differentiation. 

The splitting of Type II into separate groups was apparently 
unknown to Hintze, 646 who found anomalies in the American Type 
II and was not convinced that it was a clear-cut group. He, among 
others, also classified Streptococcus mucosus as Type III Pneumo- 
coccus, although strains of this organism were encountered that 
gave atypical reactions. 

Yu, 1567 studying fifty-one strains by the agglutination method, 



CLASSIFICATION OF PNEUMOCOCCI ill 

found at least three subgroups in Type II. Sugg, Gaspari, Flem- 
ing, and Neill 1354 in 1928 described a peculiar strain which, while 
possessing a partial antigenic relationship to typical Type III 
Pneumococcus, had distinct immunological properties of its own. 
In a later study of this same strain, Harris, Sugg, and Neill 595 as- 
certained that in rabbits this culture, related to but not identical 
with Type III, evoked better antibody (agglutinin) response than 
did Type III organisms, but in mice the Type III strain gave rise 
to a higher degree of protection against the homologous organism. 

Other serological variants were found by Clough (1919). 239 Her 
nine strains were agglutinated by Type I, II, and III antiserums. 
From the I and II serums, by absorption, two strains removed the 
agglutinins and tropins for the homologous cultures only and not 
for typical I or II organisms or for other atypical pneumococci. 
Absorption of the serums with homologous cultures removed ag- 
glutinins and tropins for all the atypical strains. The phagocytic 
and agglutinative reactions of the atypical organisms in monova- 
lent serums indicated that, in general, the strains were serologi- 
cally distinct, although in a few cases they exhibited some interre- 
lation. The special atypical monovalent serums showed no activity 
with Type I, II, or atypical Type II pneumococci. 

Another atypical strain of the heterogeneous Group IV was de- 
scribed by Pockels, 1101 who thought that its growth was sufficiently 
individual to warrant his calling the strain Pneumococcus planus. 
This organism has not been further identified. Gordon, 543 testing 
fifteen strains of Group IV by cross-agglutination and agglutinin- 
absorption, sorted them into three groups and eight separate het- 
erologous strains.* 

In England, Armstrong 19 was able to identify subgroups among 
Type II pneumococci which corresponded to Avery's IIA and IIB 
and, by means of agglutinin-absorption, divided Types I and III 
into subordinate groups of limited specificity within the main type 

* For a review of serological classification as it stood in the year 1922, and 
for a very readable discussion of the theory of antigens and antibodies, the 
reader is referred to Eastwood's 3 ^-* two papers published in that year. 



112 BIOLOGY OF PNEUMOCOCCUS 

to which they belonged. Armstrong regretted the use of the Ameri- 
can designation Type IV for the unclassified strains as being too 
narrow. Yu 1567 found Types I and III to be uniform and definite. 
In 1921, Cooper, with Mishulow and Blane, 273 published the first 
of a series of studies which were to bring a new order out of the 
confusion regarding the proper serological classification of Group 
IV pneumococci, and to establish many of the atypical subgroups 
as separate and definite types. By the agglutination method, 
checked by absorption tests when cross-agglutination occurred, 
the authors placed their fifty-three cultures in six small groups 
with thirteen unrelated strains — a total of nineteen types. 

In the next few years there were additional reports on the sero- 
logical classification of pneumococci, but only a few were of major 
importance. In 1921, Griffith 558 compared strains of his own isola- 
tion with standard type strains sent him from America by Flex- 
ner. To a 1 to 10 dilution of the monovalent type rabbit serums 
he had prepared was added an equal amount of the supernatant 
fluid from centrifuged peritoneal washings from mice inoculated 
with pneumococcal strains. It was found that the agglutination 
test was sufficient for the identification of the first three specific 
types and the author considered that the method of absorption of 
agglutinins was unnecessary. Group IV was separated into six 
types, including the American Types IIA and IIB, but these did 
not complete his classification. Griffith, by the way, like others, 
noted that agglutinating serum from horses was less selective than 
that of rabbits. 

Yoshioka (1922 and 1923), 15601 using strains of the American 
Types I, II, and III from the Hospital of the Rockefeller Insti- 
tute, and a Type I strain of German origin, was not always able 
to obtain type-specific protection in mice actively immunized with 
these cultures. The serum of immunized rabbits was type-specific in 
protection experiments, but fresh normal serum in fairly high di- 
lutions sometimes also agglutinated heterologous types. Yoshioka 



CLASSIFICATION OF PNEUMOCOCCI 113 

attributed these variations in normal specificity, such as marked 
decrease in agglutinability with homologous serum and the ap- 
pearance of agglutinability with heterologous serum, to serological 
modification brought about by growth of the organisms on unfa- 
vorable media. 

Takami (1925) 1373 ' 1375 had even less success in efforts to classify 
Japanese strains according to the American scheme, and said that 
all such attempts had completely failed in Japan. It seems highly 
improbable that specific types of pneumococci are not to be found 
in that country. The various modifications which the American 
strains underwent on artificial media may explain the unusual re- 
sults. Takami offered this fact as an explanation when he con- 
cluded that "for serological grouping of pneumococci one must 
work only with strains which have become virulent through animal 
passage, or strains which have been cultivated directly from the 
human body and have not undergone variation on artificial me- 
diums." Megrail and Ecker, 888 on the contrary, presented definite 
evidence that Pneumococcus possesses a type stability under con- 
ditions in which typhoid bacilli and other organisms show vari- 
ability in agglutination. The reliability of the methods for the 
serological typing of pneumococci is further supported by the re- 
sults of Christensen 229 and of Griffith, 558 who demonstrated the 
complete specificity of the mouse-protection test. 

In 1929, Cooper, with Edwards and Rosenstein, 272 tested 120 
strains of pneumococci isolated from cases of lobar pneumonia 
that either did not agglutinate or else reacted atypically with di- 
agnostic antiserums for Type I, II, and III pneumococci. These 
authors included Avery's IIA and IIB strains, representatives of 
various types from Pittsburgh, and the atypical III strain de- 
scribed by Sugg, Gaspari, Fleming, and Neill. 1354 Cooper and her 
associates prepared monovalent serums from rabbits and horses, 
and with serum divided the 120 strains into ten groups containing 
four or more strains each, and a miscellaneous group comprising 



114- BIOLOGY OF PNEUMOCOCCUS 

strains differing from the others, which at that time could not be 
further subdivided. These ten groups were designated as Types IV 
to and including XIII. 

Finding that therapeutic antiserums for Types I, II, and III 
had little protective power against Types IV to XIII, the authors 
prepared from rabbits monovalent antiserums of high agglutina- 
tive and protective titer for each type. The Cooper Type IV in- 
cluded an "Antitoxin" strain from the Lilly Laboratories, Robin- 
son's Group IVB strain, and one that corresponded to Griffith's 
10. These strains were highly virulent for mice, and of the nine- 
teen human cases of lobar pneumonia from which pneumococci 
were isolated, sixteen were rated as severe. Type V included 
Avery's HA and Robinson's IVE strains. All showed a tendency to 
hemolyze red cells in blood broth, and had a high initial virulence 
for mice, which however was rapidly lost. Type VI strains were 
usually less virulent for mice and their hemolytic action less 
marked than that of Type V, but more pronounced than that of 
the majority of other strains. Type VI corresponded to the largest 
group separated by Griffith from Group IV and found by him to be 
second in prevalence to Types I and II. The Type VI strains had 
such a low virulence for mice that they were not suitable for pro- 
tection tests, nor could their virulence be sufficiently increased for 
this purpose. Type VIII included Robinson's Group IVA organism 
and the atypical Type III strain described by Sugg and his asso- 
ciates. These strains agglutinated with Type III serum to such a 
marked degree that they might easily have been identified as be- 
longing to this original type. All Type IX cultures showed low 
virulence for mice. The Type X strains possessed slight or mod- 
erate virulence. The four strains designated as Type XI exhibited 
moderate hemolytic action and were of average to high virulence 
for mice, which however was quickly lost. The Type XII strains 
were moderately virulent for mice. The virulence of the Type XIII 
strains was similar to that of Type XL 

In 1932, Cooper, with the collaborators Rosenstein, Walter, and 



CLASSIFICATION OF PNEUMOCOCCI 115 

Peizer, 274 expanded the classification to include twenty-nine types 
in addition to the first three original types of Dochez and Gillespie, 
making a total of thirty-two specific types. To show how the new 
types compared with atypical strains described by other authors, 
the following list taken from the paper by Cooper and her associ- 
ates may be repeated here : 

Type IV — Pneumococcus 10 (Griffith), Group IVB (Robinson) 

Type V — Sub IIA (Avery), Group IVE (Robinson) 

Type VI— Sub IIB (Avery) 

Type VIII — Group IVA (Robinson), atypical III (Sugg, Gaspari, 

Fleming, and Neill) 
Type XV — Pneumococcus 98 (Griffith) 
Type XXI — Pneumococcus 160 (Griffith) 
Type XXII — Pneumococcus 41 (Griffith) 

The majority of the thirty-two types showed only slight cross- 
reactions; Types II and V; III and VIII; VII and XVIII; and 
XV and XXX being exceptions. Only a small percentage of strains 
isolated in New York City could not be serologically identified by 
Cooper and her co-workers. 

In the study just cited, from the majority of horses immunized 
for a period of more than a year serums with 500 to 1,000 units 
were obtained, and by the concentration of serum of lower po- 
tency, Cooper and her associates obtained preparations equal to or 
stronger than high-grade unconcentrated serums. In addition, the 
authors developed potent bivalent serums for those types which 
gave marked cross-agglutinative reactions. 

The original publication should be consulted for full informa- 
tion concerning the incidence and severity of cases due to the dif- 
ferent types, their presence in normal individuals and in spinal 
meningitis, and their virulence for mice. The original serological 
classification of Dochez and Gillespie as amplified and extended by 
the additions discovered by Olmstead and by Cooper and her col- 
leagues covers practically all strains that have been studied and is 
now tentatively accepted as standard. 



116 BIOLOGY OF PNEUMOCOCCUS 

The occurrence of many of these pneumococcal types in Ger- 
many has been reported by Silberstein (1933), 1287 who found 
twenty-one of the new types, while there were only three strains 
which could not be classified. Types XVI, XXI, XXIII, XXVIII, 
XXX, and XXXII which had not before been reported in Ger- 
many were among those identified by Silberstein. Of all the types, 
XVIII and XIX seemed to be among the more common, as had 
earlier been noted by Gundel and Schwartz.* 

That there may possibly be types beyond the present serological 
classification is suggested by the report of Christie (1934). 231 Of 
one hundred Group IV strains tested, forty-seven failed to react 
with specific serum for any of the thirty-two known types. Nine- 
teen of thirty-six cultures from healthy persons, sixteen of thirty- 
six strains obtained from convalescent carriers, and twelve of 
twenty-eight organisms isolated from patients with acute pneumo- 
coccal pneumonia could not be identified by Christie with any of 
the type-specific serums. In India, Napier and Dharmonda 943 en- 
countered among the pneumococci isolated from forty-five cases of 
lobar pneumonia and from fifteen cases of bronchopneumonia, 
strains that failed to correspond to any of the recognized types. 
Forty-six per cent of the strains studied apparently belonged to 
two types found only locally. 

Recently two strains of pneumococci were described by Smith 1296 
which the author claims constitute two new serological types. The 
organisms were isolated on separate occasions from the respira- 
tory tract of a man suffering from "chronic bronchitis." A third 
but similar strain was found to be present in pure culture in the 
lungs of an individual dying of pneumonia. The strains grew ana- 
erobically and were virulent for mice but, under the conditions 
tested, were not infectious to guinea pigs. The three strains ap- 
peared to represent two distinct serological varieties both of which 
differed from the thirty-two known types. There was no cross- 

* Quoted by Silberstein. 



CLASSIFICATION OF PNEUMOCOCCI 117 

agglutination between the two varieties and, with the exception of 
one strain, that agglutinated with Type XVI immune serum in a 
dilution of 1 to 32, the two varieties failed to be agglutinated with 
any of the thirty-two type serums. 

Strains of pneumococci which failed to correspond to any of the 
thirty-two known types were encountered in China by Wu and 
Zia, 1552 who reported that among a total of 162 strains tested, four 
failed to agglutinate with any of the thirty-two type serums. The 
unclassifiable strains possessed typical cultural characteristics and 
were virulent for mice. 

It may be expected that other types of pneumococci will in time 
be added to Cooper's list. There were still a few strains which did 
not fit into any of the thirty-two groups, and the work of the two 
Japanese bacteriologists, while leaving doubts as to the soundness 
of their conclusions, and the results obtained in England, India, 
and China present the possibility that more heterogeneous strains 
may be encountered which will be found to form additional sero- 
logical types. 

There is the further question of the pneumococci causing infec- 
tions in animals other than man. Grenier (1912) 555 described cul- 
tures from three sources, namely, guinea pigs, rabbits, and horses. 
The first two cultures from guinea pigs and rabbits were isolated 
from animals undergoing experiments with other toxic or infec- 
tious agents. The pneumococcus infecting the guinea pig was mod- 
erately virulent for mice and usually led a saprophytic existence in 
the respiratory and digestive tracts. The culture from infected 
horses did not originally kill mice but acquired virulence on animal 
passage. The observations of Grenier suggest that in veterinary 
bacteriology there may be strains, or even types, which might be 
found to have interesting pathogenic and serological relationships 
with pneumococci of human origin. The authors of the present vol- 
ume disclaim any acquaintance with the literature of that branch 
of bacteriology, so far as it concerns Pneumococcus, but studies on 



118 BIOLOGY OF PNEUMOCOCCUS 

this phase of the biology of Pneumococcus, if they have not al- 
ready been made, should be undertaken because of the additional 
information which may accrue. 

Classification According to Electrophoretic Potential 
Another criterion for classification, other than agglutination or 
protection tests, was advanced by Thompson (1931). 1396 By deter- 
mining the electrophoretic rate of migration, he grouped the sixty- 
seven strains studied into five groups. The A group included typi- 
cal Type Ill's and one Group IV strain. Group B consisted of 
typical Type I strains and a few Group IV strains. C took in Group 
IV strains, an atypical III, and two atypical II organisms. D was 
represented by a typical Type II, a few of Group IV, and one 
atypical Type I strain, while E was represented by only two 
Group IV strains. Since Groups C and D were shown not to be 
definitely distinct from each other and since the number of strains 
comprising Group E was too small to be significant, there remain 
only three large electrophoretic groups. In a second paper, Thomp- 
son 1397 presented data on the rate of migration in the electro- 
phoretic field and the isoelectric points of various pneumococcal 
strains, but made his classification somewhat unwieldy by the addi- 
tion of intermediary groups. While knowledge of the differences in 
the electrophoretic potential of different strains of pneumococci 
may be of scientific interest, the method has no advantage over the 
serological classification. 

We are now learning of yet wider deviations from special specifi- 
cations than those we have already discussed. At first sight they 
seem to confuse the definite lines of demarcation that have been 
drawn between bacterial species and the still finer distinctions that 
have been established on both chemical and serological grounds for 
types within the species. Pneumococcal cells possess polysaccha- 
rides peculiar to each type, and the carbohydrate is looked upon 
as the factor that determines the exact place of a Pneumococcus 



CLASSIFICATION OF PNEUMOCOCCI 119 

within the species. Lately' it has been found that other and appar- 
ently unrelated bacteria also elaborate complex carbohydrates, 
and that these carbohydrates both as antigens and as haptens 
exhibit immunological similarities to the polysaccharides of Pneu- 
mococcus. Puzzling as these new developments are at present, they, 
like the discovery of the transformative processes, will only lead to 
the disclosure of new biological principles. 

There is no doubt that the members of other microbial species 
and materials of vegetable origin contain polysaccharides which 
may be found to possess chemical and immunological relations 
analogous to the capsular carbohydrates of Pneumococcus but, as 
Heidelberger and Avery concluded, this type of immunological cor- 
respondence in no way invalidates the systematic classification of 
bacteria based on the more usual and general methods of species 
determination, and it is of greater immediate significance in con- 
nection with the study of problems dealing with bacteria as disease- 
producing agents than in the study of bacteria in their generic 
relationships. 

Type Determination 

The immunity reactions which form the basis of serological 
methods for the bacteriological differentiation of pneumococci are 
of inestimable importance to students of Pneumococcus and to 
clinicians. They aid in defining the relations and interrelations of 
members of this bacterial species ; they afford clues in tracing the 
spread of pneumococcal infection ; they reveal the influence of 
chemical constitution in determining antigenic specificity and im- 
mune response ; and have an eminently practical bearing on serum 
treatment. 

Whatever benefit is to be derived from the use of antipneumococ- 
cic serum depends on the rapid and accurate determination of the 
type of Pneumococcus causing the infection. While the potency of 
serum and the judgment directing its administration are of great 



120 BIOLOGY OF PNEUMOCOCCUS 

importance, it is the use of the immunologically appropriate se- 
rum and the promptness of treatment which in many cases may de- 
termine the issue for the patient. The major effort, therefore, be- 
sides increasing the accuracy of these tests, has been expended in 
devising ways of shortening the time elapsing between the collec- 
tion of the specimen and the identification of the serological type 
of the infecting Pneumococcus. 

MOUSE PROTECTION TEST 

It was Neufeld and Haendel 991 who, in 1910, realizing the im- 
portance of the delay involved in the isolation, cultivation, and 
identification of pneumococci from infected material, saw in Unger- 
mann's 1433 suggestion of testing cultures on the basis of phagocytic 
action, possibilities for reducing the time factor. Ungermann's 
plan was to inject a mouse intraperitoneally with sputum, then 
several hours later to inject immune serum, and after a further in- 
terval of an hour and a half, to kill the mouse and make stained 
smears of the exudate from the surface of the liver. When the 
serum corresponded immunologically with the culture, marked 
phagocytosis of the cocci took place. 

The use of the mouse as a vital differential medium cut short the 
time usually required by plating and subculturing and became the 
foundation of the protection test developed by Cole and his asso- 
ciates. 86 This method, which is given in detail in the Appendix, im- 
mediately came into general use, and while in the routine examina- 
tion of pneumonic material it has given place to the more rapid 
presumptive tests, it still remains the method of choice for the ulti- 
mate determination of pneumococcal types. There are certain diffi- 
culties encountered in the practice of this method, such as the 
occurrence of pneumococci of more than one type in the specimen, 
the overgrowth of Pneumococcus by other organisms in the peri- 
toneal cavity of the mouse, the occurrence of cross-agglutination 
when undiluted or slightly diluted immune horse serum is used, and 



CLASSIFICATION OF PNEUMOCOCCI 121 

the complication arising from a latent mouse-typhoid infection in 
the test animals.* 

Sutliff's 1359 experience emphasized the apparent errors which 
may occur when the testing of sputum by the mouse method is 
taken as the only diagnostic criterion. He compared the results of 
the protection test with those obtained by other cultural methods 
or, in other words, checked the type of Pneumococcus found in the 
sputum with the type or types isolated from the same patient by 
means of blood cultures, from post-mortem lung cultures, and from 
miscellaneous exudates. Among eighty-one cases in which Group 
IV strains were found in the sputum, twelve showed the presence 
of pneumococci of one of the fixed types in cultures obtained from 
the blood or lung. This error of 14.8 per cent is significant. Sut- 
liff demonstrated the advantage in performing a type determina- 
tion on cultures isolated from the hearts' blood of the test mice 
as well as on those from the peritoneal exudates. In 1,326 such 
examinations, the outcome was positive in fifty-five instances with 
the hearts' blood where the peritoneal exudate was negative. More- 
over, Sutliff at times recovered two types of pneumococci from the 
same mouse. Of 339 cases where the peritoneal exudate showed a 
fixed type, the hearts' blood of the same test animals in fifteen, 
or 4.4 per cent of these instances, yielded an organism of Group 
IV and, in five, or 1.5 per cent of cases, pneumococci of a dif- 
ferent specific type. In 862 cases in which the peritoneum of the 
mouse yielded a Type IV organism, the hearts' blood showed fixed 
types in thirty-one cases, or 3.6 per cent. Sutliff then, in all cases 
showing Group IV pneumococci in the sputum, collected and ex- 
amined a second specimen by the mouse method and here the same 
discrepancy held. Of 145 cases, twenty-two gave results on the sec- 
ond determination inconsistent with those of the first. Sutliff's ex- 
perience prompted him to advise that "When a specific type of 

* Faber,378 feeling that a substitute for mice was needed, recommended rab- 
bits, but it is feared that he failed to appreciate the economic aspects of such a 
substitution. 



122 BIOLOGY OF PNEUMOCOCCUS 

pneumococcus is obtained from the sputum by mouse test, it may 
safely be considered the cause of the disease, but when a pneumo- 
coccus which does not react specifically (type 4) is found in the 
sputum, a second examination will in approximately 15 per cent of 
the cases yield a specific reaction for one of the fixed types." 

Gundel 568 pointed out the various sources of error in the bac- 
teriological diagnosis of pneumococcal infections. He recommended 
the examination of successive specimens of sputum, mentioned the 
difficulty presented by the presence of more than one type of Pneu- 
mococcus in sputum, and explained the replacement of organisms 
of the fixed types by those of the heterogeneous "X" (American 
Group IV) by assuming that virulent Type I and II organisms 
disappeared from the sputum as the pneumonic disease progressed, 
thus allowing a predominance of pneumococci from the upper re- 
spiratory tract. 

The reports cited above are introduced to emphasize the need 
for thoroughness and care in the examination of infected material 
submitted for pneumococcal diagnosis. In some specimens, a sin- 
gle type of organism may be present and may so predominate that 
its identification by any one of the methods is a simple matter. 
When, however, pneumococci are few in number, or when variable 
results occur, the sputum should be examined by other methods 
and controlled by serological tests on cultures derived from single 
colonies. 

In 1917, Blake 128 devised a method for obviating the difficulties 
in the mouse test. He injected the mouse with sputum and, when 
infection had sufficiently progressed, washed out the peritoneal 
cavity with sterile saline solution, centrifuged the washings at high 
speed, and to one part of the sterile supernatant fluid added an 
equal part of diluted immune serum. When the organism was of the 
same serological type as that of the serum a precipitate formed im- 
mediately. Used with a large number of strains, Blake found this 
test to be consistently positive and specific for Types I, II, and 
III. The principle of the method lay in the liberation of soluble 



CLASSIFICATION OF PNEUMOCOCCI 123 

specific substance, or precipitinogen, from the pneumococcal cells 
during growth in the peritoneum of the test animal. 

CULTURE AGGLUTINATION 

In 1918, two rapid methods were announced. The urgency of 
war-time needs, the lack of laboratory facilities in the hastily im- 
provised army camps, and the shortage of mice, all called for a 
simplification and speeding up of the methods for pneumococcal 
type determination. Taking advantage of the more rapid growth 
of pneumococci in sugar broth, Avery 34 planted washed sputum in 
bouillon containing one per cent glucose and after five hours' incu- 
bation, that is, in the period of active growth of pneumococci and 
before accompanying bacteria had multiplied to any conflicting 
extent, the broth was used as an agglutinating antigen with the 
three type serums. This procedure gave a high percentage of suc- 
cessful results, besides being simple, inexpensive, and speedy of 
execution. Oliver 1026 later made the suggestion that the Andrade 
indicator be added and that inulin be substituted for dextrose in 
the Avery medium, because these modifications yielded better re- 
sults and were successful in cases when dextrose broth failed. 

An exception to the general experience with the Avery method 
was that reported by Beckler, Marden, and Gillette, 97 who had a 
number of failures with this test as compared with mouse inocula- 
tion. Since they had no control over the collection of the speci- 
mens, the work being done in a state diagnostic laboratory, the 
sputum was often mixed with saliva, which the authors thought 
might account for their unsatisfactory experience. Those readers 
desiring to learn of the operation of the Avery rapid cultural 
method in the field should consult Vaughan 1448 who gave (1918) a 
description of laboratory requirements and satisfactory results in 
army base hospitals. 

URINE PRECIPITATION TEST 

Another diagnostic, or more exactly prognostic, aid of the same 



124 BIOLOGY OF PNEUMOCOCCUS 

nature was the urine test of Dochez and Avery, 821 who had ob- 
served that in vivo as well as in vitro, Pneumococcus produced a 
soluble specific substance, and that with an appropriate immune 
serum its presence could easily be detected. Type III organisms 
formed the largest amount of the substance, Type II somewhat 
less and Type I the least. The majority of patients who failed to 
show the precipitable substance in the urine recovered, whereas the 
mortality was high among cases in which its presence was demon- 
strable. A positive test was also of diagnostic significance. 

SPUTUM PRECIPITINOGEN 

A rapid method depending upon the demonstration of pneumo- 
coccal precipitinogen was that of Mitchell and Muns (1917). 904 
They ground sputum with fine sand to disrupt the cells and release 
the soluble substance, extracted the mixture with salt solution, 
centrifuged the extract until clear, and then added immune serum 
to the supernatant fluid. The test required only an hour for its 
completion and gave clear-cut results for Type I, II, and III 
pneumococci. Those sputums which gave no reactions were classed 
as Group IV. The success of the method depends on obtaining a 
specimen rich in pneumococci. 

SPUTUM DIGESTION 

Krumwiede and Noble, 761 also responding to the demands of the 
time, sought by digesting sputum with antiformin to bring the pre- 
cipitinogen into solution, but difficulties were experienced in apply- 
ing this technique to all specimens, and the final results were often 
unsatisfactory. In a second paper, Krumwiede with Valentine, 762 
abandoned the use of antiformin and, instead, coagulated the spu- 
tum with heat. They broke up the coagulum and extracted the 
soluble antigen with saline solution in a boiling water-bath, cleared 
the extract by centrifugation, and then layered or floated the anti- 
gen over the type serums. The authors incubated the tubes at 50° 



CLASSIFICATION OF PNEUMOCOCCI 125 

to 55°, and observed a more or less opaque contact ring when the 
antigen and antiserum were of the homologous type. Here again 
there were some failures due to the quality of the sputum, but with 
the majority of specimens the method gave a rapid and accurate 
type diagnosis. 

The Krumwiede method was given a trial by Kohn 737 with a lim- 
ited number of cultures. The results, in the main, agreed closely 
with those obtained by the mouse-protection test. Because of ac- 
curacy, simplicity, and the saving of time and mice, Kohn advised 
its more general adoption. 

A somewhat unusual case was reported by Gilbert and Daven- 
port 514 which called attention to the complication presented by the 
occurrence in sputum of pneumococci of more than one fixed type. 
The Krumwiede test was negative with the three type serums. The 
culture in the Avery medium showed a faint reaction with Type I 
serum but none with that of Types II and III. A mouse inoculated 
with the sputum was dead at the end of forty-eight hours and the 
agglutination, precipitation, and cultural tests all showed the sole 
presence of Type III Pneumococcus. Direct planting of the spu- 
tum and of the Avery culture on blood plates yielded a predomi- 
nance of green-producing cocci, which culturally proved to be 
Type I organisms and S. viridans. Serologically the growth ag- 
glutinated with both Type I and III serums. 

Another avenue of approach was that reported by Loewe, 
Hirschfeld, and Wallach. 821 Instead of searching for precipitino- 
gen in the excretions of pneumonia patients, they sought precipitin 
in the blood. The blood was drawn into potassium oxalate, laked 
with ether, and added to saline suspensions of type strains of pneu- 
mococci grown on glucose-serum agar. The mixtures were incu- 
bated until a color change due to alteration of the hemoglobin ap- 
peared. Inasmuch as immune substances do not usually appear in 
the blood stream in the very early stages of pneumonia, it is diffi- 
cult to see how this method could possess any advantage over those 
aimed to detect the presence of precipitinogen in the urine or blood. 



126 BIOLOGY OF PNEUMOCOCCUS 

BILE-SOLUTION OF SPUTUM 

In 1920 and 1921, Oliver 10256 reported a method by which the 
precipitable substance of the pneumococci in pneumonic sputum 
was brought into solution by bile and the filtered material then set 
up against known antiserums. With incubation at 42° to 45°, 
when the serum and the specific substance derived from the organ- 
isms were of the same serological type, immediate clouding and 
flocculation appeared, the whole procedure requiring only about 
thirty minutes for completion. Checked against the mouse-protec- 
tion method there was full agreement in the results obtained. 

The Oliver technique had advocates in Sharp and Urbantke. 1261 
These authors in a study of forty cases found that when pneumo- 
cocci of fixed types were intermingled with streptococci and Group 
IV organisms, the presence of the former was often obscured and 
identification difficult. The Oliver method appeared to be more suc- 
cessful than the classic mouse method with such specimens. Direct 
type determination of the infecting strain in pneumococcal menin- 
gitis was rendered feasible by slightly modifying Oliver's scheme. 
The sediment from centrifuged spinal fluids was treated with so- 
dium taurocholate, the mixture shaken, and after being allowed to 
stand, centrifuged at high speed. The supernatant fluid was then 
mixed with type serums. A positive outcome was indicated by the 
rapid formation of a precipitate. The procedure could also be car- 
ried out on a microscope slide with equally definite results. 

Rosenthal and Sternberg 1175 proposed another rapid procedure 
for type determination, which consisted in digesting sputum in a 
borax-boracic acid solution and adding to the clear supernatant 
fluid of the centrifuged mixture, in separate sections on a slide, 
specific antiserums. The results by this method agreed in the ma- 
jority of instances with those obtained by the mouse inoculation 
test. 

Instead of utilizing the presence of precipitin as a therapeutic 
control, Noble 1017 described a simple, though not particularly 
original, method for determining the presence of agglutinins in the 



CLASSIFICATION OF PNEUMOCOCCI 127 

blood. The patients' serum was added to heavy suspensions of 
pneumococci. These materials used in small quantities and in con- 
centrated form gave a rapid end result, and because of its reli- 
ability Noble used the method to measure agglutinins in the blood 
of pneumonia patients during serum treatment. 

SLIDE AGGLUTINATION 

In 1929, Sabin 12<u introduced his simple rapid "Stained Slide" 
microscopic agglutination test. Briefly, the test consisted in in- 
jecting the sputum into the peritoneal cavity of a mouse, then a 
few hours later puncturing the abdominal wall, withdrawing a 
small amount of peritoneal exudate, mixing it with the diagnostic 
serums of the various types in separate drops on the same slide, 
and finally smearing, fixing, and staining the mixtures. The slide 
was examined under an oil immersion lens for evidences of agglu- 
tination. Sabin also applied this method to the determination of 
antibody in the blood of pneumonia patients for the control of se- 
rum administration, as proposed by Park and Cooper. 1057 A drop 
or more of the patient's blood was withdrawn into a capillary tube, 
and after coagulation and contraction of the clot, the tube was 
centrifuged and a minute amount of the extruded serum spread 
with a loopful of a saline suspension of a heat-killed culture of the 
type for which agglutinins were sought. The film was air-dried, 
stained for one-half minute with fuchsin, and examined micro- 
scopically. When checked against the Noble and macroscopic ag- 
glutinin methods, this test was found by Sabin to be two and 
one-half to three times more sensitive when standard diagnostic 
type serums were used for comparison. In a paper published in 
1930, Sabin 1202 gave further details and refinements of the tech- 
nique. In addition to using the method for the control of serum ad- 
ministration, he found it to be of service in detecting the presence 
or absence of active infection in man. 

Armstrong (1931 ), 22 giving Sabin credit for originating the 
method, introduced a slight modification of Sabin's technique by 



128 BIOLOGY OF PNEUMOCOCCUS 

mixing with droplets of peritoneal exudates from mice previously 
injected with pneumonic sputum loopfuls of diluted Type I, II, 
and III agglutinating serums. Instead of spreading and fixing the 
film, the material was immediately examined under a cover-slip. 
The differences in the two methods are minor and the selection of 
one or the other is a matter of personal choice. The important ob- 
servation of Armstrong's was that an increase in the size of the 
cocci appeared after the addition of homologous serum — the sig- 
nificant Quellung effect first observed by Neufeld and later made 
the basis by Neufeld and Etinger-Tulczynska 987 for their rapid 
type-determination method. A year later, Armstrong 23 published a 
supplemental report on the results obtained by his method. In 
every case the type determinations were confirmed by mouse inocu- 
lation and other methods not specified. He also reported its ap- 
plicability to cerebrospinal fluids, pus from empyemata, aural dis- 
charges, and exudates from various sources. 

Logan and Smeall 823 slightly changed the Armstrong modifica- 
tion of the Sabin method, but the change was insignificant and the 
satisfactory results the authors obtained with it might well be 
credited to the original procedure. 

Calder 196 devised another slight modification of the Sabin tech- 
nique. The cultures grown in Avery's medium were dried on a 
cover-glass, stained with a drop of gentian violet solution, and 
after the addition of diluted type serums, the preparation was ex- 
amined as a hanging drop. 

Valentine, 1444 in 1933, introduced yet another variation of the 
Armstrong-Logan and Smeall modification of the original Sabin 
method. Here again, sputum and serum samples were mixed, al- 
though in a somewhat different manner, smears made and stained 
first with carbol-fuchsin and then carbol-thionin. The bodies of 
the cocci, when the immune serum was homologous for the strain, 
stained black while everything else on the slide was red, including 
the capsule. It is difficult to discover any advantage in this tech- 
nique over the simpler methods. 









* 4* 





Figure 1 







After Neufeld and Schnitzer 1 ' 



Figure 2 



THE QUELLUNG PHENOMENON 

Figure 1. Agglutination and Quellung of pneumococci by immune serum 
in vitro. Figure 2. Pneumococci from the peritoneal cavity of a mouse 
mixed with concentrated heterologous immune serum (control). 



Is S '?. 




..*• 

m s 



• « ' v; ... 

V:. 

• , • \ 



■ i , ■ » ^ 



Figure 3 



":'<-;•■ '.t-\ 




THE QUELLUNG PHENOMENON 
Figure 3. Pneumococci from the peritoneal cavity of a mouse mixed 
with concentrated homologous immune serum. Figure i. Pneumococci 
from the peritoneal cavity of a mouse mixed with concentrated homolo- 
gous immune serum; particularly marked Quellung and mass formation. 



CLASSIFICATION OF PNEUMOCOCCI 129 

The Sabin method, either in its original or modified form, was 
for some time the one of preference. Among the many satisfied 
users of the original technique was Brown 151 of the Connaught 
Laboratories. Heffron and Varley, 602 reporting in 1932 the experi- 
ence of the Bacteriological Laboratory of the Massachusetts De- 
partment of Public Health with the Krumwiede, Sabin, and macro- 
scopic agglutination methods, preferred the Sabin procedure for 
routine examinations because of the greater number of positive re- 
sults and the shorter time required. The types as determined by the 
Sabin method were in all cases checked by agglutination of the cul- 
ture from the hearts' blood of infected mice. The simple technique 
was so readily learned by laboratory technicians that Heffron rec- 
ommended its adoption by diagnostic laboratories. 

Bullowa and Schuman, 185 for the purpose of still further reduc- 
ing the time factor when a determination of any of the thirty-two 
types was desired, proposed the pooling of type serums other than 
I, II, and III. For example, combined lot A comprised Types IV 
through VIII, while B, C, D, and E included Types IX through 
XXIII.* With these lots, diluted with saline solution in a ratio 
of 1 to 10, 1 to 15, and 1 to 25, Bullowa and Schuman made pre- 
liminary tests and, obtaining a positive result in any group, then 
tested the strain separately against the monovalent type serums 
included in that group. These groups may be arranged to fit the 
prevalence of the various types in different communities or seasons. 

At about the same time, Amoss 13 described another method. A 
sample of the patient's blood was defibrinated, the serum filtered 
through celloidin in a Coor's filter, and the filtrate centrifuged at 
high speed. The filtrate was then concentrated on a steam-bath un- 
der diminished pressure. To aliquot portions of the concentrate 
containing the soluble specific substance of Pneumococcus were 
added equal amounts of specific serum of the three fixed types, and 
during incubation at 37° in a water-bath readings were made in a 

* Two other lots, F and G, could, of course, be added to include the remain- 
ing types. 



130 BIOLOGY OF PNEUMOCOCCUS 

beam of light at five-minute intervals. In some cases definite pre- 
cipitation was noted in the homologous mixtures after five minutes' 
incubation. To the remainder of the filtrate eight volumes of 95 
per cent alcohol were then added, and, after mixing, the tubes were 
set in the ice-box over night. The flocculent precipitate was col- 
lected by centrifugation, the residue dried by heat under a stream 
of air, and after salt solution was added, the solution was dis- 
tributed into tubes for the addition of serum. The author tried still 
another method for demonstrating the presence of precipitinogen 
in patient's blood. To the serum collected as in the first procedure 
distilled water and 0.2M sodium acetate-acetic acid buffer solution 
with a pH of 4.6 was added. The mixture was then boiled until 
coagulation was complete. The filtrate from the coagulum was 
evaporated to dryness over a free flame, and heated carefully until 
the odor of acetic acid was no longer present. The resulting dry 
concentrate was extracted with sterile distilled water, the solution 
cleared by centrifugation or by filtering through paper and the re- 
sulting liquid used as a precipitating antigen against type serums 
I, II, and III. The delicacy of this method was shown by the fact 
that precipitinogen could be detected in the blood when blood cul- 
ture remained negative. 

To repeat Amoss' conclusion: "The method requires less time 
than the sputum culture or mouse method, but has no advantage 
over the sputum extract method. It is useful for typing in cases in 
which neither sputum nor urine can be obtained" ; to which might 
be added, it furnishes to those who are not content with negative 
or doubtful results one more aid in arriving at a type diagnosis. 
Amoss also saw in this method, by detection of the soluble specific 
substance in the blood, a means of judging the amount of serum 
necessary and for following more closely the results of serum ther- 
apy in lobar pneumonia. The intricacy of the procedure would bar 
its use in the busy routine of the average hospital laboratory, but 
it would be of service, where time and the facilities permitted, in 



CLASSIFICATION OF PNEUMOCOCCI 131 

estimating the degree of absorption, distribution, and neutraliza- 
tion of injected pneumococcal antibodies. 

Another variation of the precipitin method was that described 
by Sia and Chung (1932). 1271 They seeded cultures into poured 
plates of dextrose-beef-infusion agar containing, in separate Petri 
dishes, type-specific serums. After incubation, when the type of or- 
ganism and serum corresponded, a well-defined annular opacity 
surrounded each colony, an appearance which proved to be type- 
specific. The method is obviously somewhat limited in value. It re- 
quires discrimination in the quantity of inoculum as well as in the 
amount of serum to be added and since, with these disadvantages, 
it also requires twenty-four hours' incubation and large supplies of 
immune serum, there is no reason for selecting it instead of the less 
complicated and more rapid methods. In the same year, Petri 1084 
described a procedure which he used in the study of pneumococcal 
dissociation, but which, because of the recent development of sim- 
pler techniques, has not been adopted. 

QUELLUNG PHENOMENON 

The latest method to be generally used is based on the Quellung 
effect first described by Neufeld 974 in 1902. It promises to super- 
sede all the previous rapid precipitation and agglutination meth- 
ods. The technique as reported by Neufeld and Etinger-Tulczyn- 
ska 988 consists in placing on a slide, separately, three loopfuls of 
the sputum to be examined, with two loopfuls of undiluted type- 
agglutinating rabbit serum on each drop of sputum, and then the 
same amount of Loeffler's methylene blue. Each drop of the mix- 
ture is covered with a cover-glass, and examined at intervals for 
five to thirty minutes through an oil immersion lens. When the 
type of the serum and of the pneumococcus are the same, the cocci, 
stained blue, are seen surrounded by swollen capsules. The cap- 
sules are a pale greenish-gray with a characteristic ground-glass 
appearance, and are distinctly outlined by a thin, dark line around 



132 BIOLOGY OF PNEUMOCOCCUS 

which appears a halo of light. When the serum and organisms are 
of different types, the pneumococci are of their usual size and are 
barely visible. This Quellung or swelling phenomenon is a distinct 
and specific reaction between the capsular substance of the pneu- 
mococcal cell and antibody of the same type. In the same year as 
that of Neufeld and Etinger-Tulczynska's publication, Sabin 1207 
applied the method in the examination of sputum of one hundred 
cases of lobar pneumonia. The results with Type I and II organ- 
isms were found to correspond exactly to those of the mouse test. 
In two cases Sabin was able to identify Type I organism, whereas 
the results by older methods were negative. Furthermore the test 
was sometimes positive in purely salivary specimens. 

In the next year (1934), after an experience of sixteen months, 
including two months in which they extended the use of the Neu- 
feld test to all types including those from IV through XXXII, 
Beckler and MacLeod 96 confirmed the results by other methods in 
96 per cent of the examinations. They unqualifiedly recommended 
this method because of its accuracy, simplicity, rapidity, and the 
small amount of sputum required. 

Summary 

The species Diplococcus pneumoniae has been divided by means 
of agglutination and other immunological methods into thirty-two 
separate and distinct serological types. There is evidence at hand 
that points to the existence of pneumococci that may belong to 
types other than those already established. Further investigation 
may reveal some new type identities, but the present list comprises 
the great majority of the members of this bacterial species. On the 
basis of cross reactions, there appears to be some relation between 
organisms of Types II and V, III and VIII, VII and XVIII, and 
XV and XXX, but the resemblances are not sufficiently close to 
invalidate the current classification. In nature there appears to be 
a stability of the types, although, as will be shown in the succeed- 



CLASSIFICATION OF PNEUMOCOCCI 133 

ing chapter, transformation of a strain of one type into an organ- 
ism of another type can be accomplished by appropriate treat- 
ment of the culture. 

The particular serological type to which a pneumococcus be- 
longs may be determined in several ways. The agglutination, pre- 
cipitation, complement-fixation and mouse-protection tests, or 
combinations and modifications of these tests, may be applied to 
disclose the type identity of a strain, but it is safe to conclude 
that for routine needs the Neufeld method for determining specific 
types of Pneumococcus should be the one of choice. It gives an an- 
swer in the shortest time, possesses a high degree of accuracy, and 
is simple to perform. It makes for a saving in expense and elimi- 
nates the necessity of using mice. Whenever the results are not 
clear or when any doubt exists as to the type to which the organ- 
isms belong, the mouse-heart-blood or mouse-protection test should 
always be carried out for confirmation. Before hope of type iden- 
tification is abandoned, the more refined methods such as that of 
Amoss should be tried. The usual cultural procedures, carbohy- 
drate fermentations — especially of inulin — and bile-solubility tests 
are always to accompany the bacteriological diagnosis when un- 
certainty arises. For clinical diagnosis and for the direction of se- 
rum administration, as well as for epidemiological studies, labora- 
tories should be prepared to run their cultures through the series 
of the thirty-two known serological types. The procedure requires 
a supply of potent, monovalent type-specific immune serums, pref- 
erably prepared by the immunization of rabbits with standard 
type strains. With type-specific serums, accurately prepared re- 
agents, healthy laboratory animals, and careful bacteriological 
manipulations, there should be few cultures that cannot be sero- 
logically classified within the known fixed types. 



CHAPTER V 

PNEUMOCOCCAL DISSOCIATION AND 
TRANSFORMATION 

Changes in the morphological, cultural, pathogenic, and immuno- 
logical characters of pneumococci caused by various physical, 
chemical, and serological conditions in their environment; the 
transformation of the diplococci from one serological type to an- 
other; and the relation of the species to streptococci. 

The constancy of the biological characters of Pneumococcus 
depends on the conditions of its surroundings. When the con- 
ditions are favorable, the morphological and serological integrity 
of the cell remains stable. Subjected to unfavorable influences, 
Pneumococcus exhibits great lability of form and function. The 
form of the cell may pass through every stage from the typical en- 
capsulated diplococcus to one completely denuded of capsule; 
pathogenicity may be diminished from full virulence to entire ab- 
sence of infectivity ; and antigenic and serological properties may 
lose strict type-specificity and retain only the broader species- 
specificity. With the restoration of favorable conditions, the deg- 
radation process, if it has not proceeded too far, ceases and is re- 
versed, the cell again assuming its typical characters. But, what is 
still more remarkable, a degraded coccus originally derived from a 
fixed type may, under appropriate stimulation, develop the vital 
and immunological properties of a different specific type. Thus, in 
addition to natural occurrence of variation or dissociation, the 
actual transformation of pneumococcal types has been experi- 
mentally induced and, possibly, species mutation has occurred. 
Lack of knowledge of the existence of bacterial variants and of the 
factors inducing dissociation has undoubtedly caused much confu- 
sion in bacteriological diagnosis and in the interpretation of the 
serum reactions of Pneumococcus. 



DISSOCIATION AND TRANSFORMATION 135 

Early Observations of Dissociation: 1891—1921 

Bacterial variation is not a new phenomenon. It is only the 
study and the explanation of the underlying causes that are recent. 
In 1891, Kruse and Pansini 763 first called attention to changes in 
morphology, cultural characters, and virulence of pneumococci un- 
der artificial cultivation. Pure cultures freshly isolated from pneu- 
monic material were typical in appearance during early genera- 
tions but, on continued cultivation on unfavorable media, the cells 
exhibited deviations from the normal characters. The lance-shaped, 
diplococcal forms became streptococcal or even bacillary, the cap- 
sule rapidly vanished, and virulence waned. The degenerated cocci, 
when passed through susceptible animals, regained their capsules 
and virulence, and when returned to favorable media, again showed 
normal pneumococcal morphology. Kruse and Pansini, therefore, 
noted many of the features of dissociation and were aware of the 
first causes of the phenomena. 

In the next year, Arkharow 17 reported changes taking place in 
cultures cultivated in the serum of vaccinated animals. Growth 
was slow in developing and at the end of three or four days the 
cocci began to diminish in size, to grow in long chains, and to lose 
virulence. Four years later, Eyre and Washbourn 373 described the 
variations observed on continued cultivation of pneumococcal 
strains in broth. Of one strain it was said that it differed in "mor- 
phology, biology and pathogenic properties from the parent stock. 
It, in fact, represented a distinct variety, possessing practically 
no virulence, and growing luxuriantly, even at 20°C, on all the 
usual media." The first attempts to induce this variant to revert to 
its former state were unsuccessful. Then, by passage through a 
rabbit, the variant reverted to its original form. 

Hiss, Borden, and Knapp (1905) 651 encountered organisms, in- 
distinguishable in fermentative reactions from pneumococci, which 
showed variations in morphology or agglutination, and the au- 
thors considered that the cultures were temporarily or perma- 



136 BIOLOGY OF PNEUMOCOCCUS 

nently modified pneumococci, Streptococcus mucosus, or strepto- 
cocci of hitherto unrecognized types. 

A suggestive communication, because of its anticipation of later 
discoveries, was that of Rosenow 1163 published in 1910. He pre- 
sented the results of a study of seven cultures isolated from endo- 
carditis which he believed were "modified pneumococci." All the 
strains fermented inulin and produced a variable amount of green- 
ish discoloration but no hemolysis on blood-agar plates, but grew 
atypically with the development of involution forms on media con- 
taining the patient's blood. However, cultivation in normal serum 
or blood and animal passage promptly restored normal pneumo- 
coccal characters to the modified strains. Recultivation in the pa- 
tients' serum brought out the modified characters. These special 
characters varied greatly in the strains studied ; the more chronic 
the disease process in the patient from whom the serum was ob- 
tained, the more marked were the changes. This last observation 
would seem to argue for the occurrence of variation or dissociation 
in vivo — a biological process concerning which there is still some 
doubt. 

The phenomenon of bacterial dissociation, or as it was known 
in the early years of this century, variation, received little atten- 
tion until 1915 when Friel 494 reported that prolonged cultivation 
of bacteria rendered them more susceptible to phagocytosis. He 
called the process "Piantication" (fattening for slaughter) and 
observed its operation with strains of Friedlander's bacillus, Pas- 
teurella, and Pneumococcus. The same effect was produced by ex- 
posing the organisms to immune serum ; while the reverse process 
took place when "pianticated" bacteria were grown in normal se- 
rum — they regained their resistance to phagocytosis.* 

In the next year, Stryker 1348 described the variations induced in 

* Neufeld and Schnitzer credited the first demonstration of bacterial dis- 
sociation to Friel, although Hadleyss* ascribed the discovery to Baerthlein 
(1912). The phenomenon had, however, been observed much earlier by Kruse 
and Pansini (1891),763 and by Arkharow (1892),i7 and had been described in 
some detail by Eyre and Washbourn373 in 1897. 



DISSOCIATION AND TRANSFORMATION 137 

Pneumococcus by cultivation in immune serum. When virulent 
strains were grown in broth containing homologous immune serum, 
there developed variations in agglutinability, decrease in virulence, 
inhibition of capsule formation, increase in phagocytability with 
normal serum, and a change in absorptive power and in antigenic 
properties. Reversion of the changed forms to the original type 
took place on animal passage, the number of such passages re- 
quired usually varying with the number of previous serum treat- 
ments. The immune response, as measured by agglutinins, was 
slower in rabbits injected with strains grown in immune serum 
than with those cultivated in normal serum. A spread of aggluti- 
native action was evident in the ability of the serum of immune 
rabbits injected with a serum-treated Type II culture to aggluti- 
nate pneumococci belonging to both Types I and II. Type-speci- 
ficity was being lost and replaced by species-specificity. Cultures 
grown in normal serum formed capsules upon injection into mice, 
whereas those grown in homologous immune serum under similar 
conditions showed no demonstrable capsules. This loss of the abil- 
ity of Pneumococcus to synthesize the capsular substance was 
later to assume a new and broader significance. 

Later Observations of Dissociation 

SMOOTH AND ROUGH FORMS OF PNEUMOCOCCUS 

Arkwright (1921 ), 18 in studying the colony appearance of dys- 
entery bacilli grown on media containing immune serum, gave the 
designations S and R — smooth and rough — to the dissociants be- 
cause of the corresponding differences in colony topography of 
each form. Griffith, 560 in 1923, extended Stryker's observations and 
applied the letters S and R to the two forms of colonies he ob- 
served when pneumococci were grown in media containing homolo- 
gous immune serum. The S colonies have a smooth surface, and 
the cocci forming them produce the soluble specific substance in 
broth culture, agglutinate with specific serum of the homologous 



138 BIOLOGY OF PNEUMOCOCCUS 

type, are virulent for laboratory animals, and on injection into 
rabbits stimulate the production of type-specific immune sub- 
stances. The R colonies have a rough surface, and the organisms 
comprising them form no soluble specific substance in broth cul- 
ture, agglutinate atypically, and are avirulent. Cocci of the R 
colonies may revert to the S form, or they may remain stable for 
generations. Another property of the S cells is the ability to ab- 
sorb from immune serum all antibodies for both S and R forms. 
The R forms absorb only the anti-R bodies, and when injected into 
animals fail to stimulate the formation of type-specific (S) anti- 
bodies. 

Griffith considered that the R form was differentiated from the 
S by the loss of virulence and by the ability to form capsules and 
to elaborate soluble specific substance, and that the R form repre- 
sented a stage in the degeneration of Pneumococcus from the viru- 
lent, complex type of S cell to an attenuated form with a simpler 
antigenic structure. Griffith also found that degradation might not 
be permanent and that reversion could take place after animal pas- 
sage or repeated cultivation in blood broth. The author recom- 
mended for the demonstration of variant colonies an opaque 
"chocolate" agar to which red blood cells treated with chloroform 
had been added. 

Griffith looked upon the S form as the original, unchanged or- 
ganism, the R form as a variant due to unusual growth conditions. 
The degenerative action of immune serum Griffith believed to be a 
double one. He suggested as an explanation of the change the view 
that serum might disorganize the biological functions of Pneumo- 
coccus by precipitating the capsule, thus inhibiting the secretion 
of antileucocytic substances and rendering the organism tempo- 
rarily harmless, and that when pneumococci divided in the animal 
body in the presence of immune serum, the influence of the serum 
might cause progressive attenuation of subsequent generations. In 
connection with the causes for such bacterial variations, East- 
wood 344 contributed an interesting theoretical discussion. It would 



DISSOCIATION AND TRANSFORMATION 139 

take us too far afield to quote from it here, but the communication 
is recommended to those readers who desire to learn more of the 
philosophical aspects of the phenomenon of dissociation. 

In seeking a medium that would emphasize the differences in va- 
riants, Sia and Chung, 1270 " 1 by the substitution of normal dog 
blood for rabbit or horse blood in agar for plate cultures, obtained 
such marked differences in the morphology of S and R colonies 
that differentiation, they claimed, became extremely simple. With 
a moderate degree of magnification (X 28), the S colonies were 
seen to be smooth and glossy, while the R colonies, including those 
derived from Pneumococcus Type IIS, revealed a wrinkled and 
coarsely rough surface. The R colonies also exhibited strong he- 
molytic properties. Sia and Chung tested the blood of guinea pig, 
white rat, chicken, and cat, but none was so good as dog blood. 
These authors believed that the property of dogs' blood resided in 
the cellular elements rather than in the plasma, and probably was 
due to hemoglobin. 

MODIFICATIONS A, B, AND C 

Blake and Trask (1923), 129 in conducting experiments similar 
to those of Stryker, also found that growth of pneumococci in ho- 
mologous serum resulted in a marked loss of virulence, accom- 
panied by constant and distinct changes in agglutinative proper- 
ties with respect both to the character of agglutination and the 
zone of optimal reaction. The authors found the changes not to be 
a gradual alteration of all members of a culture but, instead, there 
appeared to be a comparatively rapid and complete change in in- 
dividual organisms. The variants exhibited changes not only in 
virulence and agglutinability, but also in colony appearance, by 
means of which three modifications, called by the authors A, B, 
and C, could be distinguished. 

In the same year, analogous observations were published by 
Yoshioka. 1564 Typical pneumococci underwent apparently regular 
serological modifications when maintained under unfavorable con- 



140 BIOLOGY OF PNEUMOCOCCUS 

ditions, such as surface cultivation on unsuitable media, incuba- 
tion at 39°, and too long-continued drying. The same conditions 
also led to loss of virulence. The changes noted consisted in a 
marked decrease in agglutinability with homologous serum and in 
the appearance of an enhanced agglutinability with heterologous 
serums. The modified strains were, at times, agglutinable by anti- 
streptococcic serum. The changes appeared irregularly and sud- 
denly and did not parallel the degree of decrease in virulence. An 
immune serum obtained after immunization with an atypical strain 
agglutinated only that specific variant and not the parent strain. 
In the discussion which followed the presentation of papers on 
bacterial variability before the German Association of Microbi- 
ology at Gottingen in 1924, Neufeld 979 reported a change in bile- 
solubility of pneumococcal variants, as well as in their suscep- 
tibility to optochin. In the same year, Felton and Dougherty 420 
observed that pneumococci when grown in plain broth in an auto- 
matic transferring device suffered a loss of virulence which was 
directly proportional to the change in the hydrogen ion concentra- 
tion of the medium — the more acid the medium the greater the loss 
of virulence. Accompanying the change in virulence there was an 
alteration in the behavior of the organisms toward agglutinating 
serums. Although specific, the agglutinability of the modified 
strains became greater than that of the parent organism. 

COMPOSITE CULTURES 

Amoss 12 in 1925 published an article on the composite nature of 
a pure culture of virulent pneumococci from which he derived sev- 
eral strains by the Barber single-cell technique. These were culti- 
vated in broth containing Type I antiserum, and the pure culture 
was submitted to successive transfers in bile broth and acid broth. 
Amoss reported that the virulent strain of Type I Pneumococcus, 
after being passed through 190 mice, was composed of individuals 
possessing characters differing from those of the original culture. 
A pure-line strain derived from a single cell isolated from a viru- 



DISSOCIATION AND TRANSFORMATION 141 

lent composite culture was more virulent for rabbits and less re- 
sistant to unfavorable media than was the composite strain or 
other strains similarly obtained from the same source. Amoss iso- 
lated by the plating method an avirulent strain from the compos- 
ite virulent culture which had been repeatedly transferred and 
grown in immune serum broth, bile broth, and slightly acid broth. 
Cultures of the virulent single-cell derivative, when grown in these 
media, also gave rise to the avirulent form. Heterologous immune 
serum and also normal serum did not favor the change from viru- 
lent to avirulent variants. The avirulent strains, however pro- 
cured, were all of a single sort. They formed characteristic colo- 
nies, showed no tendency to revert to the parent type, and did not 
become virulent on repeated passage through mice. Serum from 
rabbits immunized with the avirulent variants possessed aggluti- 
nins but no protective antibodies for the parent strain. It seems 
clear from Amoss' experiments that he had succeeded in effecting a 
permanent degradation of a virulent Type I Pneumococcus, with a 
loss of type-specificity, but not of species-specificity. 

The results of Reimann's 1125 study, published in the same month 
in which Amoss' publication appeared, agreed with both Amoss' 
and Griffith's observations. Reimann reported that cultures of 
pneumococci from single-cell seedings, when grown in broth con- 
taining immune serum, bile, or even normal serum, suffered a de- 
crease in virulence and loss of type-specificity. The changes might 
take place when the pneumococci were repeatedly grown in plain 
broth or on blood agar, but were due to variations in individual 
cells, rather than in the cocci of the culture as a whole. Reimann 
preferred 2 per cent unheated blood agar to Griffith's chocolate 
agar, and on this medium there appeared characteristic smooth 
colonies along with others of the rough form. Cultures from S colo- 
nies were highly virulent, had large capsules, produced soluble 
specific substance, dissolved in bile, and were strictly type-specific. 
Strains from R colonies were avirulent for mice, had no capsule, 
produced no soluble specific substance, did not dissolve so readily 



142 BIOLOGY OF PNEUMOCOCCUS 

in bile, and had largely lost their type-specificity. Single-cell cul- 
tures propagated from S colonies, after repeated transplants 
under unsuitable conditions, produced some R variants, while sin- 
gle-cell cultures from R colonies, under the same circumstances, re- 
mained constant in character. 

SPECIES-SPECIFICITY OF ROUGH FORMS 

A few months later, Reimann 1126 published these further conclu- 
sions: 

Immune sera prepared with the degraded or variant forms of pneu- 
mococci (R strains) are similar in their reaction to sera prepared with 
the protein or cell solutions of pneumococci. They contain antibodies re- 
active with the protein of all types of pneumococci, but no antibodies 
reactive with the type-specific substances. Pneumococci of the variant 
or R form, regardless of type derivation, are serologically identical and 
have the antigenic characteristics of pneumococcus protein. They evoke 
the species-specific and not the type-specific antibodies. Antipneumo- 
coccic sera produced by immunization with S strains may contain spe- 
cies-specific antibodies in addition to those which are type-specific. 
Each kind of antibody can be removed separately from these sera by 
selective absorption with the R and S strains of pneumococci. 

These fundamental observations were later to be confirmed and ex- 
plained by the discoveries of Avery and Heidelberger of the anti- 
genic chemical constituents of the pneumococcal cell. 

Takami, 1373 contemporaneously with Amoss and Reimann, added 
a few new facts about variation. His study included certain strains 
that were apparently stable in their original characters, since they 
gave rise to no variants even after two or three years' cultivation. 
There were other strains that showed a strong tendency to vary, 
and in a short time became so changed that they no longer pro- 
duced any typical colonies. In agglutinative abilities the same rule 
held true. There appeared to be no direct relation between de- 
crease of agglutinability and atypical colony formation. The only 
two characters that were closely connected were bile-solubility and 



DISSOCIATION AND TRANSFORMATION 143 

inulin fermentation. When either of these properties was lost the 
other disappeared. 

Takami 1375 followed the in vitro experiments with a study of the 
variations displayed by pneumococci propagated in the animal 
body. Rabbits, guinea pigs, mice, white rats, and house rats were 
used for this purpose, and the variants produced in these animals 
differed in agglutinative characters from the forms developed on 
artificial media. The explanation offered was that in the body the 
organisms lose their old receptors and acquire new ones. Takami 
separated five typical strains of "culture-bacteria" (pneumococci 
long grown on blood agar) into colonies that were still markedly 
agglutinable, and into others that had lost this power. The latter 
were found to be highly virulent for mice, whereas the former were 
avirulent. 

A few years later, Kimura, Sukneff, and Meyer 711 repeated the 
dissociation experiments, using broth containing 10 per cent ho- 
mologous immune serum, with subsequent cultivation of the vari- 
ants on Griffith's chloroform-blood agar and Bieling's blood-water 
agar, both of which have a laked-blood base. The results were simi- 
lar to those reported earlier, but the authors believed that they 
had demonstrated the production of other variants in addition to 
the atypical R forms with divergent cultural and serological char- 
acters. The experimental data, however, are insufficient for judg- 
ing the claim. 

For determining the true character of normal strains and of dis- 
sociants, Schiemann 1225 adopted as a criterion the possession of a 
type-specific (dominant) hapten as a prerequisite for the forma- 
tion of type-specific agglutinins and protective antibodies and also 
for virulence. For the recognition of type-specificity the essential 
considerations were, first, coarse agglutination in homologous anti- 
serum determined by the carbohydrate nature of the hapten ; sec- 
ond, the repression of cross-agglutination in heterologous serum ; 
and, third, mouse virulence. According to these standards, in 



144 BIOLOGY OF PNEUMOCOCCUS 

addition to normal and degraded R forms, Schiemann postulated 
intermediate variants which he claimed represented pseudo-types. 
The discussion was largely theoretical, and since he gave no ex- 
perimental data, it is impossible to judge the validity of his claims. 

ELECTROPHORETIC POTENTIAL OF VARIANTS 

Falk, Jacobson, and Gussin, 383 and then Falk and Jacobson, 380 
studied another criterion for variability. The authors measured 
the electrophoretic potential of Blake's variants A, B, and C from 
Type I Pneumococcus during cultivation on blood-agar slants, 
with weekly to bi-weekly transplants, over a period of one and one- 
half years. The velocities remained constant and paralleled the 
virulence of the strains. Although the authors believed that elec- 
trophoretic potential was related in some fundamental manner to 
virulence, phagocytability, agglutinability, and other serological 
characters of microorganisms, the particular variants studied 
were indistinguishable from the parent strain in these characters, 
and after a large number of generations on blood agar showed no 
evidence of spontaneous changes. The only exception to this sta- 
bility of character was a single-cell strain of variant C which re- 
verted to the A form on passage through a mouse. 

EFFECT OF CHARCOAL, YEAST, OPTOCHIN 

The variation in pneumococci appearing after growth in broth 
containing animal charcoal or dry yeast and subsequently in opto- 
chin broth, first observed by Berger and Englemann 100 " 1 and shortly 
afterward by Morgenroth, Schnitzer, and Berger, 929 was corrobo- 
rated in 1927 by Amzel. 14 Cultivation in these media gave rise to 
rough colonies, the members of which were avirulent for mice and 
exhibited diminished solubility in bile. One strain developed hemo- 
lytic properties, and another became agglutinable with antiserum 
for the fixed types. The variations observed after cultivating the 
cocci in the presence of bile were the same as those occurring in the 
Schnitzer-Berger medium. 



DISSOCIATION AND TRANSFORMATION 145 

In Amzel's 15 next paper it was reported that pneumococci iso- 
lated from pneumonia patients before optochin injections were of 
the smooth type, while the organisms cultivated after injection 
grew as rough colonies. Untreated cases yielded only smooth colo- 
nies and, in two cases repeatedly treated with optochin, the iso- 
lated culture was persistently composed of both smooth and rough 
forms. Amzel attempted to convert the rough into smooth strains 
by mouse passage but was able to effect this reversion in only one 
of three trials. 

During the 1920's there came abundant confirmation and expan- 
sion of the earlier observations on pneumococcal dissociation. Ja- 
cobson and Falk ( 1926-1927 ), 674 " 5 continuing their earlier studies, 
were able to degrade smooth strains of Blake and Trask's A, B, 
and C modifications into rough strains by growing the organisms 
in broth containing specific immune serum, although after twenty- 
three transfers the conversion was incomplete. The cultures were 
still mixtures of S and R varieties. The former continued to have 
the same virulence and electrophoretic potential, but the latter 
were reduced in both virulence and potential. Rough variants of 
the B and C strains reverted after twelve transfers in homologous 
immune serum broth, and showed the same virulence and potential 
as the original smooth organisms. In all the strains studied there 
were alterations in virulence accompanied by parallel alterations 
in electrophoretic potential and by reciprocal changes in agglu- 
tinability. Levinthal 800 also observed changes in virulence and in 
the cultural and serological behavior of a highly virulent Type I 
pneumococcus after cultivation in serum broth. He was able to 
effect the transformation of R to S forms by growth in broth at 
25° and by subsequent mouse passage. 

IN VIVO VARIATION 

Similar variations apparently taking place in vivo were de- 
scribed by Wadsworth and Sickles. 1474 Cultures isolated directly 
from the blood stream of horses undergoing immunization, or at 



146 BIOLOGY OF PNEUMOCOCCUS 

the necropsy of animals that had died as a result of the develop- 
ment of pneumococcal lesions in the heart or other organs, exhib- 
ited attenuation of virulence, loss of capsule formation, antigenic 
power and type-specificity, and changed susceptibility to phagocy- 
tosis. In the case of some of these variants the specific characters 
of the original type from which they were derived were quickly re- 
stored by one or two mouse passages. In other instances, the or- 
ganism remained atypical. 

Sickles, 1279 in a later study (1932) of pneumococcal strains that 
had become atypical in the tissues of horses undergoing immuniza- 
tion, in comparison with the typical cultures from which they were 
derived and with various other typical and atypical strains, found 
that all the organisms were bile-soluble. The maximal limits of 
growth, along with the other characters, such as limiting hydrogen 
ion concentration and peroxide and hemoglobin formation, were 
similar for the same type culture whether original, degenerated, or 
reverted. Sickles found only one strain which departed from the 
general rule and that organism grew at 42°, and survived even 
after incubation at 43.5°. No other pneumococcal strains studied 
were alive after twenty to twenty-four hours at 42°. 

That Pneumococcus may, however, retain its specific type char- 
acters when growing in the animal body was proved by Megrail 
and Ecker 888 in 1924, who injected mice and rats with suspensions 
of gum tragacanth followed by a saline suspension of pneumo- 
cocci. In these fixation abscesses the strains displayed no variation 
and no change in agglutinability. Here the conditions differed from 
those in the horses harboring pneumococci, as reported by Sic- 
kles, 1279 since the rats and mice had not been subjected to any im- 
munizing treatment, and their tissues, therefore, presumably con- 
tained no antibodies which might favor variation. 

Reimann 1128 found that R forms occurred in vivo but could dis- 
cover in his experiments no positive evidence that recovery from 
pneumococcal infection depended upon the degradation of virulent 



DISSOCIATION AND TRANSFORMATION 147 

S forms to avirulent R forms with their subsequent destruction by 
phagocytosis. In a still later study 1129 it was noticed that daughter 
colonies frequently appeared among the R variants, and in some 
instances tended to replace the typical R forms. The daughter 
strains grew in colonies with glistening surface, morphologically 
indistinguishable from genuine S colonies, although the characters 
of the bacteria comprising the daughter colonies conformed to the 
R variety. Strains of R pneumococci, which had seemed irreversi- 
ble, were apparently converted into the S form when treated by the 
method of Griffith, that is, by growth in specific immune serum. 
Reimann considered that recent experimental studies indicated 
that virulent S pneumococci might dissociate into the R form in 
vivo, that R forms occasionally could be found in the sputum of 
pneumonia patients, and also might live dormant in vivo for a con- 
siderable period of time. 

The recovery of R variants from the body has recently been re- 
ported by Shibley and Rogers. 1262 Twenty-four lung punctures 
made in lobar pneumonia patients at the time of crisis or lysis 
yielded R forms of pneumococci in all but four cases. 

DETAILS OF COLONY FORMATION 

Dawson 299 maintained that colony morphology alone could not 
be considered as a final criterion of dissociation ; it should be con- 
firmed by specific agglutination and virulence tests. While it was 
possible, by mouse passage, to accomplish a complete reversion of 
Type IIR to Type IIS, it was not possible to convert the particu- 
lar Type IR strain studied to the corresponding S form. In the 
case of a Type IIIR strain, it required twenty-eight mouse pas- 
sages to restore the variant to its original S condition. Growth of 
the same R strains in broth containing 10 per cent anti-R serum 
resulted in reversion of Type IIR on the fifth transfer, of Type 
IIIR after eight to twelve transfers, but failed to affect the Type 
IR. Dawson thought that the reversion of R to S did not depend 



148 BIOLOGY OF PNEUMOCOCCUS 

on the presence of an admixture of both forms within the culture, 
but rather that each individual R strain might or might not pos- 
sess the ability to revert. This varying tendency of R strains was 
exemplified in one experiment in which one of four other strains of 
Type IR, obtained by growing a freshly isolated Type IS strain 
in 25 per cent Type I anti-R serum, reverted to Type IS after 
forty transfers. 

The finer details of colony appearance of R and S forms inter- 
ested Paul (1927), 1067 who chose a small number of standard R 
and S strains and studied their growth on agar under a limited 
number of cultural conditions. Paul described the R colonies as 
having a rough surface, with a gradual and progressive increase in 
size over a period of several days and a tendency to remain dis- 
crete. The colonies failed to undergo rapid autolysis in early gen- 
erations and exhibited limited secondary colony formation. Methe- 
moglobin formation was present but might be replaced by slight 
hemolysis. Paul's S strains grew in rapidly developing disc-shaped 
colonies with a smooth surface which later showed irregularities. 
The colonies tended to become confluent and exhibited marked au- 
tolysis in thirty-six to ninety-five-hour cultures. In the same pe- 
riod, secondary colony formation took place. Methemoglobin for- 
mation was a constant feature. 

In a second paper, Paul 1068 gave further information concern- 
ing the conditions which affected colony formation. Under extreme 
crowding, the individual S colonies gave way to irregular, amor- 
phous, slightly elevated masses with myriads of tiny structures 
having irregular and roughened surfaces, comparable to R colo- 
nies, but on transfer to less crowded conditions they developed as 
typical S colonies. The true R colonies tended to remain discrete, 
but in dense growth resembled the S colonies under similar condi- 
tions. The effect of age on the S colonies was to increase autolysis 
and papilla formation. With the R colonies there was no autolysis, 
but roughness, opacity, and compactness became emphasized, with 



DISSOCIATION AND TRANSFORMATION 149 

papillae appearing on about the fourth day. When the blood con- 
tent of nutrient agar fell below 5 per cent, the S colonies ap- 
peared small and rough, yet were not true R colonies. The same 
effect was brought about by an alkaline reaction of the medium, 
but the original characters were restored on transplantation to a 
more favorable medium. 

In a study of the bile-solubility of Pneumococcus, Atkin 29 re- 
ported that pneumococci growing in papillae or secondary colonies 
developing on an autolyzed colony from a point inoculation on a 
thick serum-agar medium of proper reaction were insoluble and 
that susceptibility of the variants to the action of bile corre- 
sponded to the possession by the organisms of autolysin. When the 
insoluble cocci were subcultured on a fresh serum-agar slant, they 
regained autolytic properties and bile-solubility. 

Grumbach 563 also studied the details of colony formation accom- 
panying the varying degrees of pneumococcal dissociation. He dif- 
fered with Atkin, 29 but agreed with Paul that daughter colonies 
were not identical with R forms of pneumococci, because they were 
never truly granular on ascitic agar, they remained bile-sensitive, 
were fully virulent, and on transplantation developed into "bud- 
carrying" S colonies. Grumbach found that the ability to produce 
hemolysis on blood agar and in blood bouillon in forty-eight to 
seventy-two hours quite commonly ran parallel with the dissocia- 
tion phenomena. He described the characters of three virulent S 
strains isolated from pneumonic material that were not aggluti- 
nated by Type I, II, or III serums. Growing for twenty-four 
hours on ascitic agar the organisms produced the classical picture 
of pneumococcal colonies. The thickness of the peripheral ring va- 
ried, and in one case there was a "wall" formation of the type 
Buerger and Ryttenberg 169 claimed to have found solely in colonies 
of streptococci. Grumbach also described a "wing-form" colony 
which he believed to be similar to that supposed to be caused by 
phage action on streptococci, and concluded that the same colony 



150 BIOLOGY OF PNEUMOCOCCUS 

pictures could be obtained for pneumococci as for streptococci, 
but was not sure how far the bactericidal action of the body fluids 
or how far bacteriophagic action were to be considered as the basis 
for the phenomena. 

Farago (1932) 390 investigated the possible participation of bac- 
teriophagic action in the dissociative processes, but decided that it 
was not a factor. He objected to the designation R and S for dis- 
sociants, because secondary colonies were formed from virulent or- 
ganisms, whereas Griffith's R modification arose from avirulent 
strains. It is difficult to follow Farago's reasoning, but it may be 
possible that he had in mind some of the features later described 
by Dawson. 

ANTIGENICITY OF ROUGH FORMS 

Tillett 1406 turned his attention to the antigenic properties of the 
dissociated R forms. When he vaccinated rabbits by repeated in- 
travenous injections of suspensions of heat-killed R pneumococci, 
the animals acquired a marked degree of active immunity to infec- 
tion with virulent S forms of Pneumococcus I and II. (Tillett 14046 
had previously shown that a similar immunization treatment in- 
duced active resistance to Type III infection.) Furthermore, the 
whole citrated blood of the immune rabbits passively protected 
normal rabbits against infection with Type I and Type III pneu- 
mococci, but failed to confer a like protection on mice. According 
to Tillett this form of acquired resistance to pneumococcal infec- 
tion elicited by R organisms devoid of type-specificity, and exem- 
plified in animals whose serum possessed no demonstrable type- 
specific antibodies, presented features which strongly suggested 
that the underlying mechanism differed from that concerned in 
type-specific immunity.* 

RESPIRATORY CAPACITY OF VARIANTS 

Another difference in the character of S and R forms was the 

* For a full discussion and bibliography of microbic dissociation up to that 
time the reader is referred to Hadley's^ comprehensive article. 



DISSOCIATION AND TRANSFORMATION 151 

changed respiratory capacities of pneumococcal variants. Accord- 
ing to Finkle's 440 measurements, the capacities of organisms of 
Types I and II were altered during conversion from the S to the R 
form. For Type I Pneumococcus it was increased 110 per cent, 
while for Type III it was diminished by 45 per cent. In the case of 
Type II there occurred a diminution of only 16 per cent in re- 
spiratory activity. At the same time, anaerobic glycolysis was in- 
creased on the average 25 per cent each for all R forms irrespec- 
tive of type derivation, while Type I Pneumococcus, on being 
converted to the R form, lost its capacity for aerobic glycolysis. 
Pneumococcus III in passing to the degraded stage gained this 
activity, which is in accordance with the respective increase and 
decrease in respiratory activity of the two types. In order to ap- 
preciate the degree of the respiratory capacity of pneumococci, 
Finkle stated that the 2 consumption was for Type I pneumo- 
cocci thirteen times and for Type II strains thirty-four times that 
of the human tubercle bacillus (strain H 37 ). When compared with 
the oxygen consumption of animal tissues, Type II strains con- 
sumed over twenty times as much oxygen as did isolated rat kid- 
ney tissue, and almost one hundred times as much 2 as isolated 
dog muscle. 

A respiratory phenomenon connected with loss of virulence has 
been described by Sevag and Maiweg. 1258 A virulent pneumococcus 
on being transformed into its avirulent form consumes many times 
more oxygen than the parent organism, but the gain of activity is 
a temporary property. After a time, the avirulent variant degener- 
ates into a form that consumes much less oxygen than either the 
virulent or the recently derived avirulent form. The phenomenon 
may be associated with the change in the structure of the enzyme 
responsible for carbohydrate biosynthesis during the shift from 
the virulent to the avirulent state and hence may be related to cap- 
sule formation. According to Sevag and Maiweg, the addition of 
colorless, clear, blood catalase or of a small amount of sodium 
pyruvate to the culture enables the organisms to carry on their 



152 BIOLOGY OF PNEUMOCOCCUS 

respiratory functions and to maintain their reproductive capaci- 
ties and virulence for a longer period of time. 

Petrie (1932) 1084 suggested one more means for the identifica- 
tion of R and S variants. In stab cultures in agar plates contain- 
ing 5 per cent immune serum the virulent S pneumococci grew with 
a distinct halo about the colony when the organism and immune 
serum corresponded in type-specificity. The halo apparently con- 
sisted of a specific precipitate formed by the interaction of the 
pneumococcal polysaccharide and the precipitin in the homolo- 
gous serum. The R colonies, in contrast, produced only a faint and 
narrow halo after a considerable period of incubation. Serum from 
immune horses appeared to be more suitable than serum from im- 
mune rabbits for halo production. 

INTERMEDIATE FORMS 

In addition to the well-known S and R forms, Klumpen (1932) T80 
mentioned intermediate forms growing in colonies designated as 
SU and RK. In other characters, however, the strains were either 
true S or R forms. Klumpen recognized the Flatterformen de- 
scribed by Grumbach, and noted that the organisms comprising 
daughter colonies were of the smooth type. 

Still other variants intermediate between the S and R forms 
were derived from pneumococci by Blake and Trask (1933). 130 By 
growing Type IS pneumococci in homologous immune serum broth, 
the progressive appearance and disappearance of forms differing 
from both S and R cocci were observed. The forms were desig- 
nated as a, b, c, d, and e. Two of the intermediates, Type lb and 
Type Ic, were easily stabilized in pure culture. All showed an or- 
derly change in agglutinative reactions in homologous and heter- 
ologous immune serum, and also underwent a progressive loss of 
virulence for mice. Blake and Trask produced only one intermedi- 
ate form from Type IIS and none from Type HIS. 

The importance of recognizing intermediate variants in the dis- 
sociative process was emphasized by Paul 1069 of Blake's labora- 



DISSOCIATION AND TRANSFORMATION 153 

tory. He believed that two methods of inducing degradative dis- 
sociation in S forms seemed to give rise to the different patterns of 
variant production. Thus, when S forms were grown in homologous 
antiserum they became rapidly stabilized as R forms, but when S 
forms were cultivated in media containing bile, the S organisms 
displayed a greater tendency to become stabilized as c forms. Paul 
showed that during the reversion of c, d, and R forms, induced by 
growth in anti-R or plain rabbit-serum broth, intermediate vari- 
ants arose in the reverse order to that in which they appeared dur- 
ing the degradation of S forms. The intermediate variants tended 
to become stabilized as b forms, which was the usual high level to 
which these strains reverted by this method. 

A process possibly related to that studied by Blake and Trask 
was reported by Eaton, 345 an associate of Blake, who described the 
production of stable strains of Pneumococcus which underwent 
rapid lysis or failed to grow at 37°. For the strains he introduced 
the term "phantom colony" or "P-C" variants. This P-C varia- 
tion, he claimed, was a change independent of the ordinary smooth- 
to-rough variation. Eaton, moreover, made direct isolation of these 
variants from cases of human infection. 

Another apparent complication in the symbols employed to iden- 
tify pneumococcal variants is to be found in the recent papers by 
Eaton ( 1934-1935 ). 845 " 6 In addition to the phantom colony or 
P-C variants, he observed smooth variants arising in the daughter- 
colony dissociation of stock smooth strains after incubation on 
blood agar at 25°. These smooth variants, called V, and the 
smooth parent strain, termed N, from which the former were de- 
rived, had the same virulence for mice and did not differ in anti- 
genic composition as determined by agglutination, agglutinin- 
absorption, and mouse protection tests. The smooth V strains were 
stable, and while they, too, formed daughter colonies they dissoci- 
ated to rough forms much less readily than did the N or freshly 
isolated strains. The N and V strains appeared to differ in their 
capsular staining reactions, and in the ability to form methemo- 



154 BIOLOGY OF PNEUMOCOCCUS 

globin in blood. Without an actual visual comparison of these V 
variants with the principals and intermediates described by other 
authors it is impossible to assign them their proper place in the 
dissociation order. 

Further study is required before giving an estimate of the sig- 
nificance of these possibly new forms, although there is already 
much evidence to support the concept of a polyphasic cycle in bac- 
terial dissociation.* 

REVERSAL OF DISSOCIATION 

Griffith 562 was successful in reversion experiments in the animal 
body. Some R strains which had not entirely lost their soluble spe- 
cific substance readily reverted to the S form when passed through 
the mouse. The author obtained smooth colonies, with restoration 
of virulence and original serological type characters, after making 
massive injections of R strains into the subcutaneous tissues of the 
mouse. The original change from S to R forms was accomplished 
by ageing the colonies on chocolate blood agar containing horse 
serum and by cultivation in broth to which specific immune serum 
had been added. The greater the concentration of immune serum, 
the more complete and permanent was the change to the R form. 

The possibility of the reversal of the dissociation process at- 
tracted Dawson and Avery 304 who, by mouse passage, not only 
brought back to the S form seven or eight cultures of single-cell 
isolation, pure-line S strains of Types I, II, and III, but also suc- 
ceeded in causing six pure-line R strains to revert to type-specific 
forms by growing the cultures in media containing anti-R serum. f 
The authors failed in a similar attempt with a Type IR culture. As 
in mouse passage, reversion by cultural methods was accompanied 

* Rakietenins believed that a peculiar organism obtained from the peritoneal 
fluid and heart's blood of a mouse after inoculation with a highly virulent Type 
II Pneumococcus was a pneumococcal variant. It was a Gram-positive bacillus, 
bile-soluble, agglutinated with Type II serum in a dilution of 1 to 400, and also 
to a slight extent with Type I serum. The organism was not pathogenic for 
mice. Rakieten's description of cultural development of the strain from the in- 
fected fluids raises doubt as to its true pneumococcal origin. 

f Compare Soule'sisos similar results with Bacillus subtilis (1927). 



DISSOCIATION AND TRANSFORMATION 155 

by acquisition of properties of the typical S form. In the experi- 
ments, reversion was invariably toward the specific type from 
which the R form was originally derived.* 

REVERSION BY MEANS OF PNEUMOCOCCAL VACCINE 

In Griffith's experiments on reversion he introduced a new prin- 
ciple, which later was to effect still more surprising and momentous 
changes in the biological character of Pneumococcus. He reported 
that the most certain method of producing reversion was to add to 
the R culture before subcutaneous injection into the mouse a dose 
of a heat-killed culture of a virulent strain of the same type. Re- 
version from R to the S form could occasionally be brought about 
by the simultaneous inoculation of a virulent culture of another 
type when the culture had been heated for only a short period to 
60°, that is, a Type IIR strain reverted to its original condition 
when inoculated with a heated, virulent Type I culture. The Type 
I antigen appeared to lose the power to cause reversion more easily 
than the Type II antigen, the former becoming inactive after heat- 
ing to 80°, whereas the latter was still effective after steaming at 
100°. Griffith found, moreover, that the antigen of certain Group 
IV strains appeared to be closely related to that of Type II. Both 
were equally resistant to heat, and stimulated the reversion of R 
forms derived from Type II, but failed to bring about the rever- 
sion of the RI strain to its S form. 

Transformation of Type 

More surprising and important was the successful transforma- 
tion by the method of an R strain derived from one specific type 
into the S form of the same type as that of the heated culture. 
The S form of Type I was evolved from the R form of Type II 
Pneumococcus, and the S form of Type II from an RI organism. 

* Alloway (1932)8 cited Kelley as having discovered that normal hog serum 
was rich in these anti-R bodies and could be substituted for anti-R serum in 
activating the reversion process. 



156 BIOLOGY OF PNEUMOCOCCUS 

From the RI variant and from the R forms of Type II, were 
derived the clear, mucinous colonies of Type III. The newly devel- 
oped Type III strains were of relatively low virulence and fre- 
quently remained localized at the site of subcutaneous inoculation. 
A still wider shift which Griffith effected was that of a Group IVR 
strain to virulent strains belonging to Types I and II. The injec- 
tion of large doses of heated cultures of R pneumococci along with 
small amounts of living R strains never caused a transformation of 
type and only rarely produced a reversion of the R form of Type 
II to its S form. Griffith, therefore, along with his success in chang- 
ing R variants back to the original S forms with accession of viru- 
lence and specific type characters, was the first to accomplish a 
true transformation of one pneumococcal type into another. 

To degrade a pneumococcus in vitro to a form devoid of its 
original type characters and then to exalt it to its original condi- 
tion was an achievement that we had come to expect, but trans- 
forming a degenerated or dissociated culture into another form 
possessing entirely different type characters was a somewhat 
amazing performance. Even remembering the theories of earlier in- 
vestigators with their claims of species mutations, and discounting 
possible errors in their experiments, this discovery had not been 
anticipated. It was, for the first time, to supply a theoretical ex- 
planation for the many baffling problems encountered in the study 
of the spread and the invasiveness of pneumococci, and of the clini- 
cal pathology of pneumococcal infections, not to mention the 
broader bearing on the many riddles of microbiology. 

Neufeld and Levinthal 994 also were able to reproduce Griffith's 
transformation phenomena, but by another procedure. They first 
dissociated virulent, type-specific strains by growing the organ- 
isms in broth containing sterile animal organs (spleen, heart, kid- 
ney, and liver of rabbits). The degraded R variants were then 
injected subcutaneously into mice with killed S pneumococci. Neu- 
feld and Levinthal thus converted an avirulent Type IR pneumo- 
coccus into a virulent Type IS organism, and with the addition of 



DISSOCIATION AND TRANSFORMATION 157 

a killed Type IIS strain obtained a typical IIS pneumococcus. Not 
all the R variants could be reverted. 

Somewhat less success in this respect attended the efforts of Rei- 
mann. 1129 The R strains evolved by immune serum-broth cultivation 
were as a rule irreversible, only one of many strains passing back 
to the S form of Type I or over to Type III, the reversion depend- 
ing upon the type of the heated culture used. No transformations 
to Type II occurred, although in one instance it appeared that a 
heated IIS culture induced the reversion of the R strain to the 
Type IS prototype. Reimann obtained positive reversions of typi- 
cal R forms from pneumococci of Types I or II when he inocu- 
lated the R strains subcutaneously into mice with heated S cul- 
tures of Types I, II, and III. The living IR culture plus heated 
Type IIS vaccine gave Type IIS pneumococci ; IIR became IS or 
HIS, depending upon the type of heated culture used. Both Types 
IR and IIR, when inoculated with heated cultures of homologous 
type S forms, frequently reverted to the respective prototypes. 
These seemingly bizarre biological changes were, therefore, becom- 
ing a routine laboratory performance. 

Baurhenn's 93 efforts at reversion (1932) were more fruitful than 
Reimann's. By subculturing R strains with homologous and heter- 
ologous vaccines consisting of heat-killed cultures, he changed the 
R strains into their original S forms and to the S form of a differ- 
ent type. Baurhenn inclined to Griffith's view that all pneumococ- 
cal types possess a common basic form. The basic form, under the 
stimulation of the activating principle, responds by acquiring the 
properties of the activator. Baurhenn claimed to be the first to 
have produced transformation within Group X (Group IV) as 
well as the transformation of a fixed type (I, II, or III) into a 
specific type of Group X. This feat is, of course, entirely possible, 
and from what we already know of the phenomena of transforma- 
tion, there is no reason to doubt that similar changes may occur 
in the case of all the known types of pneumococci. 

Dawson (1928) 2 " confirmed and expanded Griffith's observa- 



158 BIOLOGY OF PNEUMOCOCCUS 

tions. He found that type-specific S pneumococci could be trans- 
formed from one specific S type into another specific S type 
through the intermediate stage of the R form; that R forms of 
pneumococci, derived from any specific S type, might be trans- 
formed into S organisms of other specific types by injecting mice 
subcutaneously with small amounts of living R strains together 
with heated vaccines of heterologous S cultures. The S vaccines 
could be heated for fifteen minutes between 60° and 80° and still 
remain effective in causing R forms derived from heterologous S 
types to revert to the type of the vaccine ; S vaccines heated fifteen 
minutes at temperatures between 80° and 100° were not active in 
causing R variants derived from heterologous S types to revert to 
the type of the vaccine; S vaccines heated between 80° and 100° 
could cause Type IIR and Type IIIR variants to revert to the 
original S type ; S vaccines of any type, including Type I, heated 
for fifteen minutes at 80° to 100° would no longer cause Type IR 
strains to revert to their original S type; S vaccines heated for 
periods as long as two hours at 60° were effective in causing R 
forms derived from heterologous types to revert to the type of the 
vaccine employed. Dawson successfully converted a single-cell R 
strain derived from a Type HIS pneumococcus into a Type HIS, 
a Type IS, and a Group IVS organism. On the other hand, every 
attempt to produce transformation of type in vivo failed. 

TRANSFORMATION BY VACCINE AND ANIMAL INOCULATION 

In 1930, Dawson and Sia 305 announced the transformation of a 
Type IIR into a Type HIS pneumococcus. The conditions neces- 
sary for the reversal were minimal amounts of the R culture, the 
addition of the heated activating culture, incubation for longer 
than the conventional period, and the inclusion of small amounts 
of anti-R serum and of blood broth. When the activating organ- 
isms were heated for fifteen minutes at 100°, they lost their capac- 
ity for inducing transformation, although suspensions heated for 
four hours at 60° or for fifteen minutes at 80° were still effective. 



DISSOCIATION AND TRANSFORMATION 159 

Filtrates of vigorously growing cultures and of heat-killed suspen- 
sions of S organisms were inactive, as also were suspensions of S 
organisms disrupted by freezing and thawing, with subsequent 
heating for fifteen minutes at 60°. But when suspensions of S or- 
ganisms were first killed by heating for fifteen minutes at 60° and 
then frozen and thawed, they were highly effective. In a more de- 
tailed communication, the authors gave the additional information 
that transformation of type could be induced by the use of small 
amounts of S vaccine, and that while the transformative process 
was brought about most readily by employing anti-R serum in the 
culture medium, it might be accomplished without the presence of 
the serum. 

Transformation of one S form to the S form of a different type 
without any apparent development of intermediate stages was de- 
scribed by Dawson and Warbasse 306 in 1931. The original culture 
was a virulent, single-cell isolation of Type II Pneumococcus. One 
drop of a 10" 6 dilution of the culture was seeded into a medium 
containing homologous immune serum together with large quanti- 
ties of Type III pneumococcal vaccine. The cultures were incu- 
bated at 37°, and at the end of forty-eight hours streaked plates 
showed, in the majority of instances, numerous Type III with 
some Type IIS colonies. No R colonies were observed. From the 
experiment Dawson and Warbasse inferred that a type-specific S 
pneumococcus can be transformed into other type-specific S pneu- 
mococci by growth in homologous immune serum in the presence of 
heterologous vaccine. Although the conditions of the experiment 
were unfavorable to the development of R forms, the authors 
thought it was probable that the organism nevertheless passed 
through this intermediate stage during the transformation. 

In 1931, Sia and Dawson 1272 reported that R cultures possess- 
ing slight degrees of R stability were most suitable for transforma- 
tion experiments in vitro. The authors also sought a soluble prin- 
ciple in cultures subjected to the action of bacterial enzymes 
liberated in old broth cultures and during mechanical disruption of 



160 BIOLOGY OF PNEUMOCOCCUS 

young bacterial cells. Trials with the solutions, with the superna- 
tant fluid from an S vaccine, the filtrate from an S vaccine, puri- 
fied soluble specific substance, and the filtrates of actively growing 
S cultures, all gave negative results. 

ISOLATION OF THE TRANSFORMATIVE PRINCIPLE 

Alloway (1932) 8 evidently was more successful than his prede- 
cessors in obtaining the transformative principle from the pneumo- 
coccal cell. With filtered extracts of virulent S strains of Types I 
and III he converted a Type IIR strain into S organisms of the 
same specific type as that of the cells extracted. The author stated 
that the constituents of the extract supplied an activating stimu- 
lus of a specific nature in that the R pneumococci acquired the ca- 
pacity of elaborating the capsular material peculiar to the organ- 
isms extracted. 

In the next study (1933), Alloway 9 prepared active and spe- 
cific extracts by dissolving S pneumococci in sodium desoxycholate 
solution. These cell-free extracts were as potent as the intact cocci 
in causing R forms to assume new type-specific characters. With 
an extract of Type III Pneumococcus he was able to convert a 
Type IIR variant almost regularly and abruptly into the smooth 
form of Type III. Alloway then purified the extracts by removing 
a considerable amount of inert material by charcoal adsorption 
and reprecipitation of the adsorbed extracts with alcohol or ace- 
tone. The stimulating principle passed through Berkefeld filters 
without loss of strength if the reaction of the solution was alka- 
line. The substance was resistant to heating at 60° for thirty min- 
utes but was appreciably affected at temperatures of 80° or over. 
The purified extracts apparently had suffered no loss of potency 
and caused a more prompt transformation than did the original 
solutions. An unexplained observation was the fact that in no in- 
stance could the transformation be effected without the addition to 
the culture-extract mixture of blood serum or of ascitic or pleural 
fluid. 









o 










r » 



A 









Courtesy of Dr. M. A. Dawson 



MUCOID, SMOOTH, AND ROUGH PHASES OF PNEUMOCOCCUS 
a. Mucoid colonies ; twenty-four hours on blood agar. X 32. b. Smooth 
colonies ; twenty-four hours on blood agar. X 32. c. Rough colonies ; 
twenty-four hours on blood agar. X 32. d. Smooth colony with Rough 
"outbursts"; nine daj-s on blood agar. X 32. e. Mucoid, Smooth, and 
Rougli colonies ; sixteen hours on blood agar. The Smooth colony shows 
a fine granulation or stippling. X 32. f. Mucoid organisms from 
twenty-four-hour colony on blood agar. Hiss capsule stain. X 1350. 
g. Smooth organisms from twenty-four-hour colony on blood agar. 
Gram stain. X 1350. h. Rough organisms from eighteen-hour colony 
on blood agar. Gram stain. X 1350. 



DISSOCIATION AND TRANSFORMATION 161 

Dawson Classification 

A dissociation form, other than the S and R forms, was de- 
scribed in 1934 by Dawson. 303 It appeared to be a mucoid variant 
of Pneumococcus and was strikingly different from the two main, 
accepted variants. Dawson intimated that the terminology of bac- 
terial dissociation should be changed to include the M form. 

In later communications (1934*), Dawson 302 " 3 gave many more 
details of the several stages of pneumococcal dissociation. He 
showed, first, that the change from the typical, virulent form to 
the degraded variant was not a simple direct S — » R conversion, 
but that the dissociative process consisted of several phases. In 
this cycle there were three outstanding stages represented by dis- 
tinct difference in colony appearance and morphology, and here he 
departed from the orthodox concept of the S and R forms. 

At first reading, Dawson's discussion and proposals are a little 
bewildering. He makes the apparently radical suggestion that the 
old designations smooth and rough be changed and the term "mu- 
coid" be introduced into the terminology as it applies to Pneumo- 
coccus ; thus, S would become M ; R would become S ; and the new 
form would be R. His revelation of the intricacies of the dissocia- 
tive phenomena and the proposed change in terminology are apt to 
cause some confusion in minds accustomed to the accepted order 
of dissociative nomenclature. But an unprejudiced and painstak- 
ing study of the facts and his recommendations serves to dispel 
some of the doubts raised in a cursory reading of the text. Daw- 
son's contentions were founded on the appearance of a new variant 
during the cultivation of an R form of Pneumococcus originally 
derived from a Type IIS culture. When this strain was diluted, 
thinly streaked on blood-agar plates, and incubated several days 
at 37°, many of the colonies showed evidence of a variety of sec- 
ondary growths. The following is taken from Dawson's description 
of the S -» R transformation. 

For convenience the evolution of the R variant may be described in 
several stages although it is emphasized that the process is both gradual 



162 BIOLOGY OF PNEUMOCOCCUS 

and continuous. In the first stage, which may suitably be termed R 1 , the 
colonies present more or less the general appearance of smooth (old ter- 
minology, "rough") colonies but the surface is more coarsely stippled. 
The constituent organisms are more or less typical pneumococci show- 
ing a tendency to staphylococcal grouping and occasional swollen or 
club forms may be seen. In the second stage, R 2 , the colonies present a 
still rougher appearance and the outline may appear slightly irregular. 
This irregularity frequently becomes quite pronounced after several 
days' growth. The bacteria in this stage are much more pleomorphic 
and are frequently elongated in an extreme lanceolate manner. They 
still retain Gram's stain. In the third stage, R 3 , the surface of the col- 
ony becomes exceedingly rough and the margin quite irregular. The 
contour of such colonies still remains convex but less so than the origi- 
nal S form (old terminology, R form). The organisms constituting such 
colonies present a bizarre morphological picture. Pointed diphtheroid 
elements arranged in a fashion suggesting broken twigs may be ob- 
served, with scattered long, bizarre, rod forms which are partially 
Gram-positive and partially Gram-negative. At this stage of develop- 
ment the morphological picture can scarcely be recognized as that of 
pneumococcus. The fourth stage, R 4 , can only be defined with some 
difficulty. It would appear that the growth is now in a stage of con- 
siderable flux and several types of colonies and morphological elements 
may be produced. Some of the colonies present an appearance similar to 
that just described while others resemble more closely the pure R form 
(R»). 

The morphology of the organisms in the R 4 stage is difficult to de- 
scribe because of their extreme pleomorphism. In addition to coarse and 
irregular coccal forms there may appear elongated Gram-negative rod- 
like structures exhibiting irregular Gram-positive areas. A great va- 
riety of other morphological elements may also be present. 

Dawson described the R — » S change in which intermediate forms 
of the type seen in the S — > R change were not observed, and then 
gave a detailed description of the biological characters of this new 
variant. From the description a few of the more important data 
may be selected. The organism was bile-soluble, of low virulence for 
mice, agglutinated in normal saline solution, and failed to elabo- 
rate soluble specific substance. The variant was not peculiar to the 



DISSOCIATION AND TRANSFORMATION 



163 



Type IIR strain, and single organisms of one individual strain 
also possessed the capacity to dissociate into the new form. 

Dawson then pointed out certain discrepancies in the character- 
istic features of the S and R forms as described by Griffith for 
Pneumococcus and those described by Arkwright for the colon- 
typhoid-dysentery group, and which have been accepted by the 
majority of bacteriologists as the chief distinguishing features of 
the smooth and rough forms of many bacterial species. He fur- 
ther drew attention to the fact that certain attributes of Ark- 
wright's S and R forms do not appear in Pneumococcus while 
other new distinctions did not have a place in Arkwright's original 
descriptions. Dawson has portrayed these differences in termi- 
nology in a diagram which, although as yet unpublished, was 
kindly loaned to the authors. 



1 


COLON, TYPHOID, 
DYSENTERY 


PNEUMOCOCCUS 


OTHER 
ARKWRIGHT OBSERVERS 


GRIFFITHS 


DAWSON 


NOT KA 

DESCRIBED IVI 


s 


M 


2 


s s 


R 


S 


3 


R R 


NOT 
DESCRIBED 


R 



Courtesy of Dr. M. A . Dawson 



RELATIONSHIPS OF MUCOID, SMOOTH, AND ROUGH COLONIES 

OF BACILLI OF THE COLON-TYPHOID-DYSENTERY 

GROUP AND PNEUMOCOCCUS 



164 BIOLOGY OF PNEUMOCOCCUS 

When Dawson compared the salient characters of the three 
pneumococcal variants (S, R, and the new M form) with the mu- 
coid, smooth, and rough forms of members of the colon-typhoid- 
dysentery group and the smooth and the two rough forms of the 
Friedlander bacillus, the inconsistency in the use of the terms 
smooth and rough became convincingly apparent. On a basis of 
colony appearance, morphology, growth in plain broth, stability 
in salt solution, and of virulence and type-specificity, the smooth 
form of Pneumococcus and of Friedlander's bacillus conforms to 
the mucoid form of members of the colon-typhoid-dysentery group ; 
the rough form of Pneumococcus and the R x form of Friedlander 
are similar to the smooth form of bacilli of the enteric group ; while 
Dawson's new variant and Julianelle's R 2 form of the Friedlander 
bacillus agree with the rough form of the colon-typhoid-dysentery 
bacilli. 

In order, therefore, to bring these terms in agreement, to con- 
form — with an addition — to the designations of Arkwright, and to 
establish a uniform and logical terminology for the dissociants of 
all bacterial species, Dawson would change the terms now used for 
the variants of pneumococci as follows: Mucoid or M would re- 
place the present smooth or S ; smooth would be substituted for the 
former rough (R x form of Friedlander bacilli) ; while rough or R 
would be applied to the new variant described by Dawson and the 
R 2 form of Friedlander's bacillus. 

There is no doubt that such a reversal of the accepted terms 
would cause confusion and meet with opposition. It cannot be de- 
nied that this change would be especially disturbing to the present 
correlation between the classification of dissociation forms and 
immunological behavior, but that does not necessarily preclude the 
possibility of a new and perhaps a deeper insight into the parallel- 
ism between the phenomena of variation and antigenic specificity. 
This proposed change recalls the confusion that followed the revi- 
sion of the designations of blood groups, but that change has not 
only been endured but the new terms are now generally accepted as 



DISSOCIATION AND TRANSFORMATION 165 

useful and logical. There is much to be said both for and against 
Dawson's proposal and so it may be permissible to turn to one who 
speaks with authority on this important subject of bacterial dis- 
sociation. Hadley's opinion expressed in a letter written in 1933 to 
Dawson was in part:* 

Making a decision regarding the proper course to pursue in changing 
the nomenclature now employed for designating the phases of the pneu- 
mococcus, in favor of the symbolization which your studies thus far 
seem unquestionably to justify, might easily depend on how fully an in- 
vestigator has in mind the details of dissociative variation as a phe- 
nomenon observable in all bacterial species, and how clearly he can 
perceive the parallel trends in such variations, — as opposed to a limited 
outlook on the one species in which he may be especially interested. 

If bacteriology were limited to the study of a few species, or to the 
Pneumococcus, it would make little difference what the observed phases 
were called, because no generalizations would be involved, and the 
phase symbols would possess no significance for bacteriology as a 
whole. A, B and C, or X, Y and Z would serve the purpose. . . . 

The desirability of adjusting the difficulty in the Pneumococcus situa- 
tion, and of doing it without delay, is the more to be recommended in 
view of the increasingly wide recognition that the same or analogous 
phases exist in numerous other species. The facts are now becoming so 
extensive and well grounded that they are offering, for the first time in 
the history of bacteriology, a basis for the formulation of general laws ; 
and for making possible a certain kind of "predictability," as I have 
perhaps already demonstrated to you. To this extent pure bacteriology 
is beginning to take on the aspects of a real science — a compliment 
which (to my mind) it has scarcely been appropriate to offer in the 
past. 

To facilitate this highly gratifying trend it stands to reason that all 
who work with the problems of variation should keep in mind the dual 
significance of their results, and make possible a correlation of their 
own results with those of others; also to make quick and decisive cor- 
rections when such are clearly in order. To label as a smooth a Pneu- 
mococcus phase that is demonstrated to be a mucoid, or to label as a 
rough a phase that is clearly a smooth, may do little harm to those 

* The authors appreciate the courtesy of Doctors Hadley and Dawson in 
granting permission to include portions of this letter here. 



166 BIOLOGY OF PNEUMOCOCCUS 

whose work lies chiefly in this species. But such a continued policy can 
only render increasingly difficult important comparisons with other spe- 
cies, and work havoc with the interests of those who are seriously at- 
tempting to discern some law and order in the affairs of the bacteria. 
Further advance in this direction can take place, according to my view, 
only if bacteriologists become sufficiently keen to recognize the true na- 
ture of the phases they employ, and sufficiently independent to "call a 
spade a spade," whenever recognized as such, regardless of politics, tra- 
dition or social etiquette. . . . 

It might also be in the back of your mind that the splendid work of 
some of your associates on the chemical aspects of dissociation would 
suffer from any change in terminology made at this late date. I am ab- 
solutely convinced to the contrary. In reality I believe that the incen- 
tive to extensions of their results to many other bacterial species would 
be a direct and immediate outcome, through establishing a recogni- 
tion of the most appropriate culture phase to be employed in such 
studies. . . . 

It is therefore my opinion that a frank recognition of the present 
incongruities of the situation will not detract from, but facilitate in 
wide measure, researches in the important field opened up years ago by 
Drs. Avery, Dochez, Heidelberger and their collaborators. 

Dawson believed that before making such a radical change in the 
accepted terminology of pneumococcal variants it would be well to 
ascertain if similar variants could be demonstrated in Streptococ- 
cus haemolyticus. From the latest study by Dawson, 303 it would 
seem that he succeeded in dissociating that organism into three 
main variants, which in their manner of colony formation and in 
morphology correspond closely with the three main variants of 
Pneumococcus. The mucoid and smooth forms appeared and, by 
cultivation of the streptococci on blood agar and by repeatedly 
picking and transplanting material from the roughest marginal 
areas, Dawson was able to develop the extremely rough type of 
colony which he had obtained with pneumococci, representing the 
R variant. 

As Dawson said, "evidence is rapidly accumulating to show that 
the phenomenon of bacterial variation in a wide variety of bac- 



DISSOCIATION AND TRANSFORMATION 167 

terial species fits into a more or less orderly pattern." This pat- 
tern, besides fitting bacilli of the colon-typhoid-dysentery group, 
the types of B. friedlanderi, and probably the streptococci, would 
bring order in the arrangement of the many variants of pneumo- 
cocci that have been described under a wide diversity of terms. 
Thus, the modifications A, B, and C of Schnitzer and Berger, 
Blake and Trask's intermediates Type I a, b, c, d, and e, Wads- 
worth and Sickles' atypical strains, Reimann's daughter-colony 
variants, the "wall" type of Buerger, the Flatterformen of Grum- 
bach, possibly the P-C or phantom colonies and the smooth N and 
the smooth V types of Eaton, the variants of Kimura, Sukneff, 
and Meyer, the atypical rough forms from budding colonies re- 
ported by Paul, the SU and RK dissociants of Klumpen, and of 
course the R and S forms of Griffith, and the new variant of Daw- 
son might conceivably be arranged in accordance with the general 
pattern and would all either fall into the chief places designated by 
Dawson's M, S, and R or into the spaces between these predomi- 
nating forms. 

The scheme of Dawson, therefore, revolutionary as it may seem, 
merits further consideration and should be subjected to additional 
experimental trial before it is rejected or finally accepted. 

These discoveries concerning the variability of Pneumococcus 
are full of new meaning to the bacteriologist, biochemist, immu- 
nologist, and particularly to the physiologist. They prove that 
Pneumococcus has the potential ability to synthesize simple sugars 
into diverse, complex, and highly individual polysaccharides. When 
the conditions of the surroundings are entirely favorable, this 
metabolic process operates uniformly. The end products are al- 
ways of the same molecular composition and configuration, and 
are highly distinctive of a given serological and biochemical type. 
When, however, the forces of the environment are inimical, the 
function of carbohydrate synthesis is retarded, the cell produces 
less and less of the distinguishing capsular polysaccharide, and the 
cocci lose their capsule, virulence, and strict racial identity. If the 



168 BIOLOGY OF PNEUMOCOCCUS 

unfavorable conditions continue, this particular metabolic activity 
ceases or is suppressed and the organism degenerates into a harm- 
less coccus, devoid of any specialized earmarks — a sort of bac- 
terial maverick. If the exposure to these untoward conditions is 
sufficiently protracted, the function is apparently permanently 
lost, but if the exposure ceases before this stage is reached, the 
cell retains the latent power to elaborate its original, individual 
capsular carbohydrate, and all that is needed to revive this power 
is the restoration of a satisfactory environment — either in culture 
or in an animal — or else the activation that comes from an encoun- 
ter with immune bodies specific for its own degraded form. Living 
under such conditions the type-less coccus gradually returns to its 
former distinctive state. 

These discoveries, moreover, have disclosed another and aston- 
ishing activity of the organism. When stimulated by some un- 
known constituent of fully functioning pneumococcal cells, this 
latent metabolic function of the degenerated coccus develops a new 
property, and instead of building up capsular carbohydrates of 
the former kind, the degraded cell now synthesizes polysaccharides 
of the same chemical constitution and specific type as those of the 
strains supplying the activating stimulus. The once degraded or- 
ganism becomes then a virulent pneumococcus, but with all the spe- 
cialized characters of its foster strain. Having lost its original 
features it regains a new type identity. 

The cycle of degradation, regeneration, and type transforma- 
tion presents so many fascinating phases that one is strongly 
tempted to speculate on the various factors concerned in this ex- 
traordinary evolution. The basic ability to elaborate these various 
specific capsular carbohydrates is always ready to respond to ap- 
propriate stimulation unless the cells have gone too far down the 
path of degradation, and is evidently common to all pneumococci. 
The direction which the transformation takes is determined wholly 
by the nature of the stimulus, and it is the identity of this factor 
which still remains unrevealed to us. It apparently exists only in 



DISSOCIATION AND TRANSFORMATION 169 

cells exercising all their special functions, and seems to be a nor- 
mal constituent of the cell and not a product of katabolic processes. 
Whether such transformations ever take place in the animal 
body, in health or in disease, and if they do what causes bring 
them about, together with the yet broader problems of the origin 
of various types and the influences which established their differ- 
ent biological identities, are all questions that are attracting in- 
vestigators in this branch of science. Whether this fundamental 
function of Pneumococcus can be so perverted as to bring about 
the transmutation of this organism into one of a different species 
is a problem which has been attacked in a more general way. 

Transmutation of Species 

The mutability of members of the bacterial tribe Streptococ- 
caceae has long been a moot question. From time to time there have 
appeared reports of the change of a pneumococcus into a strepto- 
coccus, and even of a swing through the whole cycle from virulent 
Pneumococcus to Streptococcus viridans to Streptococcus hae- 
molyticus and back to Pneumococcus. But, in these later days of 
refined bacteriological and immunological technique one has been 
inclined to look somewhat askance at such claims. The idea has, 
however, persisted, and what was looked upon as a mere notion is 
now becoming so much more than a hypothesis that there are those 
who would accept this metamorphosis as an accomplished fact. 

There is no call to recite at any length the accounts of the early 
experiments. Some were based on crude, faulty methods which al- 
ways raise doubts as to the purity of the cultures the pioneers 
studied. Disregarding claims resting solely upon morphological or 
cultural phenomena, it is better to confine the discussion to re- 
ports, with a few exceptions of historical interest, that have been 
published since the development of modern bacteriological and 
serological technique. In 1891, Kruse and Pansini, 763 by trans- 
planting forty-six strains of pneumococci on media unfavorable to 
growth, developed eighty-four varieties that exhibited differences 



170 BIOLOGY OF PNEUMOCOCCUS 

in character all the way from typical Diplococcus lanceolatus to 
Streptococcus pyogenes. The authors stated that the relation of 
pneumococci to streptococci was clearly evident, and that the ori- 
gin of these bacterial species was a single, probably saprophytic, 
streptococcal form. 

There the matter rested until 1907, when Buerger and Rytten- 
berg 169 described an organism isolated from a case of puerperal 
pneumococcemia which, although originally failing to ferment 
inulin and exhibiting streptococcal characters, developed into a 
typical pneumococcus after animal passage. The observation led 
the authors to study a number of cultures isolated from human 
exudates and blood, and with these strains they observed charac- 
ters typical of streptococci which, however, gave way to pneumo- 
coccal characters after propagation in mice. Buerger and Rytten- 
berg concluded : 

The tendency of pneumococci of the streptococcus cultural type as 
well as those which have been converted to the normal variety, seems to 
be toward a gradual degeneration which manifests itself in the assump- 
tion of permanent streptococcic features. Such organisms can then no 
longer be differentiated from streptococci. 

In 1909, Rosenow 1162 made the statement that strains of Strep- 
tococcus viridans isolated chiefly from the blood in cases of sub- 
acute endocarditis and obtained also from the throat and other 
sources might by animal passage take on the properties of typi- 
cal pneumococci, and hence designated them as "modified pneumo- 
cocci." Rosenow also claimed that during a study of autolysis of 
pneumococci in salt solution and of the effect of sodium oleate and 
bile on virulent pneumococci he had observed transformation of 
the strains into hemolytic streptococci. The statement appears to 
be conservative when compared to Rosenow's 1170 description in 
1914 of the various transmutations accomplished within the Strep- 
tococcus-Pneumococcus group. He told of converting by cultural 
methods twenty-one strains originally isolated as hemolytic strep- 



DISSOCIATION AND TRANSFORMATION 171 

tococci from cases of erysipelas, scarlet fever, puerperal sepsis, 
arthritis, and tonsillitis, as well as from cows' milk, into Strepto- 
coccus viridans; of changing three similar strains into S. viridans 
and typical pneumococci, and one into Streptococcus mucosus as 
well. Seventeen strains isolated as S. viridans, chiefly from the 
blood and tonsils in cases of chronic infectious endocarditis, and 
two strains from cows' milk were converted into pneumococci while 
two of the strains became S. mucosus. Ten of the viridans cultures 
were made to take on the cultural and morphological characters of 
hemolytic streptococci, in two of which the pathogenic powers 
were shown to be those of hemolytic streptococci, while one strain 
was converted into a hemolytic streptococcus, into S. viridans, and 
then into a pneumococcus. 

Rosenow claimed to have converted into hemolytic streptococci 
eleven strains isolated as pneumococci from sputum, blood, and 
the lung in pneumonia and from human empyema fluids and Cole's 
Type I and II strains, while seven cultures took on the features 
of S. viridans. The streptococci derived by animal passage from 
three of the pneumococcal strains were alleged to acquire all the 
essential features of the streptococci of rheumatism, and two or- 
ganisms were said to have been converted into hemolytic strepto- 
cocci, the streptococci of rheumatism, S. viridans, and back again 
into Pneumococcus. 

Rosenow further alleged that the transformation of some of 
these strains, checked in a few instances by single-cell isolations, 
was found to be complete by every test known. The tests included 
the study of morphological features, the demonstration of cap- 
sules, and observations on fermentative powers, solubility in bile 
and in saline solution, the behavior toward the respective broth- 
culture filtrates (Marmorek's test), the specific immunological re- 
sponse as manifested by the appearance of opsonin and agglutinin 
in antistreptococcic and antipneumococcic serum, and the more or 
less specific pathogenic powers of the various organisms. 



172 BIOLOGY OF PNEUMOCOCCUS 

In summary Rosenow wrote: 

The changes observed have frequently the characteristics of true mu- 
tations because they appear suddenly, under conditions more or less ob- 
scure and because the newly acquired properties persist unless the or- 
ganisms are again placed under special conditions. A pre-mutational 
stage seems to be necessary because the same strain will not yield mu- 
tants when placed under what seem to be identical conditions at differ- 
ent times. The underlying conditions which tend most to call forth 
changes are, first, favorable conditions for luxuriant growth and then 
unfavorable conditions — under stress and strain. This seems to call 
forth new or latent energies which were previously not manifest and 
which now have gained the ascendency and tend to persist. This may 
hold true in vivo also. This fact makes it difficult to obtain mutations 
outside of the body with highly virulent strains, because they die before 
there is opportunity for the organisms to adjust themselves to the new 
conditions. It explains why injection into cavities makes for greater 
changes than intravenous injections of moderately virulent organisms. 
Apparent mutations in animals have been observed almost exclusively in 
closed cavities, such as joints and pericardium, and here mostly when 
the tissues of the host were gradually getting the upper hand and the 
organisms were being destroyed. The mutations in vitro may be spoken 
of as "retrogressive" and those in animals as "progressive" because evi- 
dences of a vigorous vegetative life are diminished whereas in the latter 
they are usually increased. 

The results and conclusions of Rosenow have been transcribed in 
some detail because they represented such a wide departure from 
established belief. The announcement was greeted with much skep- 
ticism. Such sudden and wide shifts from one to another suppos- 
edly fixed species appeared to violate biological laws, and it seemed 
that some artifact must have been responsible for the remarkable 
transformations. Nowhere in the literature, with the exceptions to 
be described, have references been found which duplicate or sub- 
stantiate Rosenow's results. 

Wolff (1923), 1534 in a long theoretical discussion of pneumococ- 
cal mutation, suggested that the members of the large tribe Strep- 
tococcaceae, from pure saprophytes to true parasites, in spite of 



DISSOCIATION AND TRANSFORMATION 173 

all differences, were really linked together. He claimed to have ob- 
tained mutations by gradual adaptations of the organisms to the 
host. The attempts met with many failures which were explained by 
saying that if the organism was too weak it died in the host, and if 
too virulent it killed the host before any accommodation had taken 
place. Wolff asserted, however, that in three cases he had trans- 
formed Streptococcus viridans from endocarditis lenta into Pneu- 
mococcus. The organism became bile-soluble, optochin-sensitive, 
developed a capsule, fermented inulin, and was lethal for mice. 
Evidence of bacterial mutations of any kind coming solely from in 
vivo experiments is to be weighed with caution. 

Neufeld 979 in a discussion already cited on microbic variability, 
recalled an observation he had made ten years previously on the 
original "Pneumococcus I" of Neufeld and Haendel, which had 
been preserved by drying and storage in a dessicator. One mouse 
inoculated with the culture produced a strain growing in chains, 
insoluble in bile, but virulent for mice, and with all typical strepto- 
coccal properties. At first Neufeld thought he had made a mistake 
in the material he injected, but a similar experience of Schiemann's 
convinced him that a mutation had actually taken place. Coming 
from anyone less eminent than Neufeld, this single, isolated obser- 
vation would be disregarded. 

In the following year, Morgenroth, Schnitzer, and Berger 929 an- 
nounced that by special methods they had been able with regular- 
ity to transform pneumococci into streptococci.* Their medium 
contained dead yeast cells or animal charcoal which had adsorbed 
optochin. The altered strains became insoluble in sodium tauro- 
cholate, were avirulent for mice, and were resistant to optochin. 
Modification A represented the first stage in the transmutation. 
The organisms retained the majority of their pneumococcal char- 
acters, but were more resistant to optochin and more sensitive to 

* Stankaisi2 called attention to the fact that these authors had omitted men- 
tion of similar work published by Elschnig and Ulbrich, and by Kraupa from 
the German Eye Clinic at Prague. 



174 BIOLOGY OF PNEUMOCOCCUS 

rivanol than were cocci of the original stock. In Modification B, 
the colonies, made up of A after growing in optochin, resembled 
those of S. viridans. The cultures contained long chains of round 
cocci, which were bile-insoluble and were very resistant to the pneu- 
mococcidal action of optochin. Modification C developed after fur- 
ther growth on artificial media or in animals, and occasionally 
after growth in an optochin medium. The C variants corresponded 
to Streptococcus haemolyticus, they produced more or less he- 
molysis on blood agar, were bile-insoluble and optochin-fast, but 
sensitive to rivanol. The progressive changes did not always take 
place or follow the A-B-C sequence. In twenty-nine experiments 
with fifteen strains, twenty-two trials produced modifications A 
and B, and of these strains seven were transformed into modifica- 
tion C. 

Berger and Englemann 100 continued similar mutation experi- 
ments and alleged to have demonstrated Modification A in five 
specimens of sputum and one of pleural exudate obtained from 
pneumonia patients before the disappearance of fever. The strains 
were then converted into Modification B by allowing a fairly high 
concentration of optochin to act upon them. Berger and Engle- 
mann also claimed that the complete transformation could take 
place in the human organism. To support the claim the authors de- 
scribed the development of glistening Type III colonies along with 
a few strongly hemolytic streptococcal colonies on a blood-agar 
plate upon which pneumonic sputum had been spread. The organ- 
ism, after the first mouse passage and three culture generations, 
developed into a green streptococcus ; after a second direct mouse 
passage both pneumococci and hemolytic streptococci appeared, 
the latter partly reverting to Pneumococcus after two culture gen- 
erations. The original hemolytic streptococci after three culture 
generations became green streptococci and after four culture gen- 
erations reverted to pneumococci. This cycle, like Rosenow's, seems 
almost too rapid and direct to be credible. 

In another communication, Berger and Jakob (1925) 102 returned 



DISSOCIATION AND TRANSFORMATION 175 

to earlier experiments on the development of B and C modifica- 
tions. During animal passage of short duration, the changes were 
less marked, since the authors reported only a transient loss of 
virulence. Berger and Englemann 101 in the next year reported the 
mutation of a strain of Type III Pneumococcus through the in- 
termediary A modification to a green streptococcus. As in their 
former experiments, the agents necessary for the transformation 
were dry yeast-broth and serum-broth containing one five-thou- 
sandth part optochin. Wirth 1523 believed that Streptococcus mu- 
cosas represented a mutation from Pneumococcus, but he failed in 
his attempts to prove it. 

In yet another paper Berger with Silberstein 103 described the 
inulin-fermentative power of the variants. The results are difficult 
to understand. Of ten strains of pneumococci, four showed merely 
a reddening of the inulin medium without coagulation, while two 
failed to display any action on inulin. The authors then classed 
the latter strains when tested with optochin with Modification A. 
Of the cultures of Modification B, obtained from pneumococci, but 
otherwise behaving as green streptococci, two retained the ability 
to ferment the carbohydrate. The strains were comparable in their 
behavior toward inulin to some thirty viridans strains. Of the lat- 
ter, five exhibited a marked action on inulin, and four others gave 
slightly positive reactions. 

Reimann, 1127 repeating the experiments of Morgenroth, Schnit- 
zer, and Berger, claimed, however, that the R cultures so derived 
were still pneumococci, since the strains were bile-soluble and 
autolyzed with greater readiness than did streptococci. The immu- 
nological reactions of the variant pneumococci derived by Morgen- 
roth's method, moreover, were identical with those of R pneumo- 
cocci derived by various other means. When one considers the 
atypical action of the Berger strains on inulin and the author's 
omission of serological tests, one is inclined to accept Reimann's 
interpretation as the correct one. 

Heim and Schlirf, 633 likewise, were unable to verify the work of 



176 BIOLOGY OF PNEUMOCOCCUS 

Morgenroth and his collaborators, yet Silberstein, 1286 who quoted 
these authors, by the aid of optochin in vitro, claimed to have ex- 
perienced no difficulty in carrying a Group IV pneumococcus 
through the successive stages of Modification B (green Strepto- 
coccus) to Modification C (virulent hemolytic Streptococcus) and 
then from this form to a Type I pneumococcus of low virulence. 
Paul 1070 was another to join the newer school which believed that 
the gap between pneumococci and streptococci could be bridged by 
these methods. He produced bile-insoluble dissociants and to him 
they appeared to be indistinguishable from certain strains of 
Streptococcus viridans. 

Gorander (1930) 542 also stated that he had transmuted cultures 
of Streptococcus viridans into bacterial forms that in every re- 
spect were identical with the type-specific pneumococci of human 
origin, except that the strains were not agglutinated by antipneu- 
mococcic serum. The defect would seem to be a vital one. The cul- 
tural changes were accomplished by repeated cultivation on blood 
agar and by short mouse passages. According to Gorander, after 
the third short (four-hour) mouse passage, hemolytic streptococci 
appeared. Following five twenty-four hour incubation periods in 
mice, the organisms resembled pneumococci. The variants had cap- 
sules, were soluble in sodium taurocholate, and were moderately 
virulent for mice. The pneumococci so obtained, after repeated 
growth of this passage culture in artificial media (alternating 
bloou agar and broth), were retransformed "into a bacterium of 
perfect Streptococcus viridans type." 

Gorander claimed further to have transformed Streptococcus 
viridans and Type I and Type II pneumococci into forms which he 
considered to be their original state, "since they were absolutely 
equal culturally, biologically and serologically in all respects." 
The homologous antiserum agglutinated both strains, and "the 
bacteria absorbed not only their homologous but also heterologous 
agglutinins from both sera." Gorander's further conclusions were 
so heterodox that they are quoted here: 



DISSOCIATION AND TRANSFORMATION 177 

Finally single cell cultures originating in their time from a single cell 
of a pneumococcus have been examined with regard to the degree of 
dissimilarity which such cultures can eventually show. These experi- 
ments gave the result that two pneumococcus cultures, obtained from 
the same cell, can show much greater dissimilarities than two cultures 
obtained one from a typical Streptococcus viridans and the other from a 
typical Streptococcus lanceolatus. . . . Thus Streptococcus viridans 
and Pneumococcus lanceolatus seem to be different forms of the same 
bacterium, and the specific agglutinability, which Pneumococcus lan- 
ceolatus shows when grown from the human body and which has been 
taken as a base for the so-called type classification, is only an occa- 
sional character. 

Summary 

Virulent pneumococci of all the known serological types, upon 
encountering unfavorable physical, nutritional, or other biochemi- 
cal conditions during growth or storage, undergo marked changes 
in virulence, in ability to elaborate capsules, in colony develop- 
ment, and in their immunological characters. In studies on the dis- 
sociation phenomena displayed by pneumococci, a great variety 
of aberrant coccal forms have been observed which are intermedi- 
ate between the typical, virulent form and the thoroughly de- 
graded, atypical form. So many variants with such a diversity of 
biological characters have been described and so many designa- 
tions have been given to the intermediate forms, that it is difficult 
to gain a clear conception of the significance of the many phases 
of pneumococcal dissociation. In order to bring order out of this 
chaos and to make the nomenclature applied to pneumococci uni- 
form with that employed in naming the variants occurring in the 
case of other bacterial species, it has been proposed to change the 
terminology now in use. Mucoid or M would replace the present 
smooth or S ; smooth would be substituted for. rough ; while rough 
or R would apply to a recently discovered variant. Whatever the 
fate of the proposal, the alterations in character which may be 
induced in pneumococci by appropriate means constitute one of 
the most important features in the biology of the species. 



178 BIOLOGY OF PNEUMOCOCCUS 

During the dissociative process antigenic action may vary from 
one of strict type-specificity to one merely of the broader species- 
specificity. Degraded forms may, if the degenerative process has 
not been complete, regain all their original morphological, cul- 
tural, and immunological characters. Regeneration can be accom- 
plished by rejuvenating the strain by passage through a suscep- 
tible animal, by cultivation in media containing an antiserum 
produced by immunization with the degraded forms, or through the 
stimulus afforded by heat-killed virulent cultures of an homologous 
type. Degraded variants, moreover, can by the action of devital- 
ized, virulent pneumococci, actually be transformed into pneumo- 
cocci of types entirely different from those from which the variants 
were derived and identical with those of the cultures stimulating 
transformation. The nature of this transformative or mutative 
principle is still unknown, but it is probable that it is a constituent 
of the pneumococcal cell and not an extracellular product of its 
metabolism. 

The broader transmutation of Pneumococcus into Streptococ- 
cus and of Streptococcus into Pneumococcus has been advanced as 
a biological possibility. Experiments have been described in which 
it was alleged that this transmutation took place. Not only has it 
been claimed that both virulent and degraded pneumococci were 
converted into avirulent Streptococcus viridans, but the organisms 
were said to have become virulent hemolytic streptococci, while the 
streptococcal forms have been further changed into pneumococci. 
Such radical departures from established theory require the clos- 
est scrutiny of the evidence advanced and of the accumulation of 
new and confirmatory facts before they can be accepted. 



CHAPTER VI 

PATHOGENICITY OF PNEUMOCOCCUS : 
EXPERIMENTAL ANIMALS 

The ability of Pneumococcus to infect animals of different species 
under natural circumstances or when experimentally introduced 
by diverse routes into the animal body; the morbid manifestations 
following pneumococcal infection in animals; virulence and other 
factors influencing inf ectivity of pneumococci. 

Pneumococcus is incapable of producing disease in animals of 
some species, while individual creatures exhibit different de- 
grees of resistance to pneumococcal invasion. Variations in sus- 
ceptibility or in resistance may be conditioned by special differ- 
ences in anatomic structure, in genetic heritage, or in physiological 
function, while individual differences may be due to both intrinsic 
and extrinsic factors affecting the physical state of the animal be- 
fore or at the time of inoculation. 

Susceptibility of the Animal Host 

THE RABBIT 

Rabbits are prone to develop spontaneous pneumococcal infec- 
tion of the respiratory tract, contracting the disease from simi- 
larly affected guinea pigs or from other rabbits.* Recovery from 
the infection may be followed by the carrier state during which the 
animal may serve as a potential source of infection to other stock 
animals, while the presence of pneumococci in the nasopharynx 
may constitute a confusing factor when the animal is used for ex- 
perimental pneumococcal infection. 

Next to the mouse, the rabbit is the most susceptible of labora- 

* This is not an uncommon laboratory experience and among the references 
at hand is that of Sanderson,! 218 w ho wrote of the spontaneous death of a rab- 
bit from Pneumococcus following a supposedly air-borne infection in the labo- 
ratory animal room. 



180 BIOLOGY OF PNEUMOCOCCUS 

tory animals to pneumococcal infection. Susceptibility is greatest 
for strains of Type I pneumococci, less for Type II organisms, and 
still less for those of Type III. It should be remembered that later 
studies have shown that while many strains of Type III pneumo- 
cocci are avirulent for rabbits, there are others of the same type 
that are highly pathogenic. 

It will be recalled that in the early days of bacteriology, Pas- 
teur, 1065 " 6 Sternberg, 1816 " 8 Vulpian, 1453 and Claxton 237 produced a 
fatal septicemia in rabbits by the subcutaneous injection of human 
saliva containing, as we now know, pneumococci. With the excep- 
tion of the intact skin, rabbit tissues present no barriers to the in- 
vasion of virulent pneumococci ; the organisms, no matter by 
which avenue introduced, soon reach the blood stream and, when in 
sufficient numbers, cause the death of the animal. 

Bacteriemia is the predominant manifestation and pneumonia 
develops only when the organisms are implanted in the lung by way 
of the respiratory tract. The weakness of the rabbits' defense 
against subcutaneous, intraperitoneal, or intravenous pneumococ- 
cal inoculation is due, in part, to the comparative inability of the 
leucocytes of the animal to engulf the invading cocci (Tongs, 
1922 1416 ). Even when injected into the cisternal cavity of the 
brain, as reported by Stewart (1927), 1322 septicemia rather than 
meningitis ensues. As a rule, therefore, after artificial inoculation 
with pneumococci, infection, unless it be by the intradermal or in- 
tratracheal route, tends to become systemic and not localized. 

Subcutaneous inoculation. Neufeld (1901 ) 973 spoke of a pro- 
gressive inflammation following the injection of pneumococci into 
the subcutaneous tissues of the ear of the rabbit. When death did 
not follow, necrosis was observed, and the infection appeared to 
Neufeld to be similar to erysipelas, although Fraenkel, who had 
previously observed the same effect, had not so considered it. 
Cooper 276 found that the mucous membrane of the buccal surface 
of the rabbit's cheek was susceptible to infection, but the inocula- 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 181 

tion in reality was a subcutaneous one, since it was necessary to 
injure the membrane by scratching it before infection took place. 

Intravenous inoculation. The rapid and frequently fatal infec- 
tion resulting from the injection of pneumococci into the circula- 
tory system has often been utilized for testing the specific resist- 
ance of rabbits after various immunizing treatments. Among the 
many references, there might be mentioned the observations of Til- 
lett (1927) 1403 " 4 who, in attempts to stimulate the production of 
immune bodies, discovered differences in the pathogenicity of cer- 
tain Type III strains for man and for rabbits. Some of the strains 
isolated from human sources, despite the possession of large cap- 
sules and high virulence for mice, exhibited low virulence for rab- 
bits. The intravenous injection of the Type III strains avirulent 
for the species produced a non-fatal bacteriemia which, however, in 
its course differed from that caused by non-encapsulated, rough 
forms of pneumococci. 

Intradermal inoculation. In 1928, Goodner 525 " 6 described the 
acute and often fatal infection developing in the rabbit following 
the introduction of Type I Pneumococcus into the skin. After the 
injection of a small quantity of a broth culture of the organism 
into the skin at the midline of the abdomen, within eight to twelve 
hours there appears a local lesion, consisting of a swollen, edema- 
tous area which may spread until the whole midabdominal region is 
involved. The development of the lesion is accompanied by an 
abrupt rise in temperature and invasion by the cocci of the blood 
stream. A varying proportion of the animals so treated spontane- 
ously recover and, as a result of the infection, may become tempo- 
rarily immune. Goodner pointed to the analogy between the nature 
of the localized and subsequent systemic infection arising after in- 
tradermal inoculation of the rabbit with Pneumococcus and that of 
lobar pneumonia in man. Since it does not come within the province 
of the present volume to discuss, except in a cursory way, the path- 
ological processes caused by pneumococcal infection, the reader 



182 BIOLOGY OF PNEUMOCOCCUS 

is referred to the original communications of Goodner, 525 " 7 ' M1 
Rhoades and Goodner, 1136 and others who have described the inti- 
mate details of the phenomena. 

Kolmer and Rule 748 employed the intradermal method to test the 
resistance of rabbits induced by previous immunization with pneu- 
mococci ; Goodner, Dubos and Avery, 536 and Goodner and Du- 
bos,° 35 for studying the effect of the polysaccharide-splitting bac- 
terial enzyme in infection with Type III Pneumococcus ; while 
Goodner, 527 Watson and Cooper, 1492 Powell, Jamieson, Bailey and 
Hyde, 1105 Sabin, 1208 Gelarie and Sabin, 510 and Curphey and Ba- 
ruch 293 applied the Goodner technique in determining the immuniz- 
ing action of specific immune serum and other agents. 

Inoculation by way of the respiratory tract. Tchistovitch 
(1890) 1381 was apparently the first to study the effect of Pneumo- 
coccus when introduced into the trachea of the rabbit. The diplo- 
cocci caused only a feeble, local inflammatory reaction with little 
phagocytosis. In 1915, Kline and Winternitz 728 described the con- 
ditions necessary to produce lobar pneumonia in rabbits. The 
catheter must be inserted as deeply as possible into a bronchus, 
and the culture fluid must be injected with considerable force in 
order that the organisms may be introduced into the alveoli. 

Permar 1082 has described in detail the manifestations appearing 
after the intratracheal injections of cultures of Type I Pneumo- 
coccus. He concluded that experimental pneumonia in the rabbit 
begins as an acute inflammatory reaction. The severity of the re- 
action increases from the trachea and bronchioles and is greatest 
in the bud-like alveoli arising from them, in the alveolar ducts, 
atrea, and alveoli. The process begins in the bronchus and invades 
other tissues by peripheral extension leading to coalescence. Acute 
interstitial pneumonia in the rabbit develops early as a result of 
acute lymphangitis arising in the peripheral lymphatics ; then the 
process extends to both pleura and hilum. Permar suggested that 
septicemia might arise as the result of the direct involvement of the 
vascular walls, or it might possibly be due to the passage of organ- 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 183 

isms through the nodes at the hilum and thence through the tho- 
racic duct into the circulatory system. The author believed that 
the process was, in its essentials, comparable to that occurring in 
spontaneous pneumonia in human beings, the chief difference being 
a more intense interstitial involvement in the experimental disease. 

Through the insufflation of the lung with cultures of pneumo- 
cocci of Types I, II, and III, and of Group IV, Gaskell (1925) 500 
reported that organisms of Group IV possessed a greater patho- 
genicity for the rabbit than those of Type I which, in turn, were 
more invasive than those of Type II, while the Type III culture 
employed was the least virulent of all. 

Stuppy and Falk (1931) 1352 found that intrabronchial insuffla- 
tion of rabbits with cultures of pneumococci of uniformly high 
virulence gave rise to bronchopneumonia which, with septicemia 
and a generalized distribution of cocci in the lungs, usually caused 
the death of the animal in two to five days. In some animals there 
was acute inflammation of the interstitial tissue of the lung, with 
perivascular and peribronchial lymphangitis. Suppurative bron- 
chitis and pleuritis were only occasionally seen. On the whole, the 
pulmonary lesions induced in rabbits by strains of Type I, II, and 
III pneumococci of the same virulence were quite similar, while in- 
dividual virulence rather than the serological type of the culture 
employed appeared to be the important factor in establishing in- 
fection. In a study of the effects of the inhalation of pneumococci, 
Stillman 1336 observed that, following the spraying of rabbits with 
cultures of virulent Type III cultures, the organisms tended to re- 
main in the lungs for a considerable period of time without invad- 
ing the blood stream. When once the organisms had reached the 
blood, a fatal septicemia resulted. The course of events was in con- 
trast to that ensuing after the similar administration of Type I 
and II strains. Organisms of Types I and II frequently entered the 
circulation, but in such instances, only a relatively small number of 
the animals died. 

Another organ of the rabbit possessing little or no resistance to 



184 BIOLOGY OF PNEUMOCOCCUS 

infection with Pneumococcus is the eye. Tchistovitch (1890), 1381 
on introducing the organism into the anterior chamber, found that 
the aqueous humor, instead of being antagonistic to the cocci, 
served as a medium for their development. According to Neufeld 
and Schnitzer, injection of virulent strains of pneumococci under 
the conjunctiva or cornea leads to a severe infection of the eye, 
which is followed by systemic infection. The same result attended 
a similar injection of mouse blood containing pneumococci (Gins- 
berg and Kaufmann, 1913 517 ). 

Chilling and wetting, age, breed, weight, and diet as factors in- 
fluencing susceptibility. The great variability in the behavior of 
rabbits toward pneumococcal infection, whether naturally or arti- 
ficially acquired, may be due to either or both internal or external 
conditions. Among external causes Kline and Winternitz (1915) 728 
studied the influence of cold, alcohol, ether, and bromine on rab- 
bits infected intrabronchially with Pneumococcus. The agents ap- 
peared to predispose the animal to the development of bronchitis 
and even bronchopneumonia, but the results were not conclusive. 
In experiments in which rabbits were given intrabronchial inocula- 
tions of virulent pneumococci, Stuppy and Falk 1352 found that wet- 
ting and chilling the animals failed to lower resistance to invasion 
of the injected pneumococci, but exposure to cold appeared to ren- 
der the test animals more susceptible to spontaneous infection. 

Freund 484 observed the difference between the reactions of young 
and adult rabbits to intradermal inoculation with virulent pneu- 
mococci — confirming the susceptibility of immature individuals 
earlier reported by Kruse and Pansini. 763 In adult rabbits injected 
with virulent pneumococci extensive inflammation developed at the 
site of infection, but bacteriemia and death occurred in relatively 
few of the animals. Younger animals failed to develop such an ex- 
tensive inflammatory process and succumbed to bacteriemia. The 
ability to respond to inoculation with an energetic, local reaction 
constitutes a barrier which apparently develops with growth of the 
animal. 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 185 

In an investigation of the physiological variables responsible for 
the lack of uniformity in the behavior of rabbits to intradermal 
inoculation with Pneumococcus, Goodner 532 concluded that, since 
normal rabbits lacked any form of specific antipneumococcal anti- 
bodies, resistance to intradermal inoculation in combination with 
passive immunization with specific immune serum was determined 
by the physiological condition favorable to the utilization of pas- 
sively conferred specific antibodies, which in turn depended upon 
the weight and white blood-cell count of the rabbits. Animals, 
therefore, that are physiologically mature possess an advantage 
over animals less mature and with a lower cell activity. 

Another factor affecting the resistance of rabbits to infection is 
vitamin deficiency. Greene 554 reported that rachitic rabbits showed 
a greater morbidity and mortality from intranasal inoculation 
with Type I Pneumococcus than did normal controls. Differences 
in the susceptibility of rabbits of diverse breeds has also been 
noted ; hence, in the selection of rabbits for comparative or quanti- 
tative tests on the virulence of strains or types of pneumococci or 
for measuring the potency of immune serum, consideration should 
be given to the breed, age, weight, and diet of the test animals. 

THE GUINEA PIG 

The comparatively low and variable susceptibility of guinea 
pigs precludes their use for many purposes in experimental studies 
on Pneumococcus. The susceptibility of the guinea pig to pneumo- 
cocci administered by inhalation was first demonstrated by Neu- 
feld and Ungermann 10012 in 1912. The inoculation, in some cases 
but not regularly, produced a slow pneumonic process. When the 
culture was injected directly into the lung, the animals developed 
acute infection, dying in one or two days from pleuropneumonia or 
bronchopneumonia. 

The intrapleural injection of pneumococci into guinea pigs may 
cause a pleuropneumonia, as shown by Kolmer. 741 Neufeld and 
Ungermann 1001 " 2 and also Engwer 365 reported similar successful in- 



186 BIOLOGY OF PNEUMOCOCCUS 

fection after the injection directly into the lung of pneumococci 
cultivated by several animal passages. 

One objection to the use of these animals is their liability to de- 
velop spontaneous pneumococcal infection, to become carriers, and 
thus to transmit infection to other laboratory animals. Such an 
outbreak was reported in 1922 by Gheorgiu, 512 but no mention was 
made concerning the type to which the infecting organism be- 
longed. The presence of pneumococci as secondary invaders in an 
epidemic among guinea pigs and mice caused by B. bronchisepticus 
was observed by Keegan. 700 Branch 147 in 1927 reported the pres- 
ence of pneumococci of Group IV in all of thirty-six guinea pigs in 
a laboratory epidemic. The organism failed to agglutinate with 
specific immune serum for the first three fixed types, but by its pro- 
tein fraction appeared to be related serologically to strains of 
pneumococci of human origin. The infection took the form of 
otitis, enlargement of the spleen, lobular and even true lobar pneu- 
monia. According to Bruckner and Galasesco as well as to Chevrel 
and Ranque,* spontaneous epidemics sometimes begin with septic 
abortion of the guinea pigs. 

The presence of pneumococci in the nares of apparently normal 
guinea pigs and of guinea pigs affected with snuffles, and the oc- 
currence of natural epidemics of pneumococcal infection in ani- 
mals of this species, was investigated by Neufeld and Etinger- 
Tulczynska 984 " 5 in 1931. The organisms responsible for natural 
infections, including those found in a previous investigation of an 
outbreak in another colony of animals, when tested by means of 
the Quellung phenomenon, proved to belong to Type XIX. The 
strain was only slightly virulent for mice and guinea pigs. The 
authors found further that animals surviving the intranasal im- 
plantation of pneumococci of Types I and XIX, as a rule, become 
carriers of the respective strains. 

Neufeld and Etinger-Tulczynska also noted wide variations in 
the susceptibility of the animals to natural infection from pneumo- 

* Quoted by Neufeld and Sehnitzer. 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 187 

cocci and, while cold, avitaminosis, and pregnancy had previously 
been shown to diminish resistance of guinea pigs, the authors con- 
cluded that higher susceptibility was apparently of a more obscure 
nature and was associated, as earlier suggested by Uchida, 1432 with 
basic or temporary variations in the disposition of individual ani- 
mals. A possible explanation for some of the constitutional differ- 
ences in the susceptibility of guinea pigs is to be found in the com- 
munication of Nicholls and Spaeth (1922), 1005 according to whom 
there is a definite correlation between pigmentation and resistance 
to infection. White-coated, pink-eyed guinea pigs, probably pure 
albinos, were found to be far more susceptible to a given Type I 
culture than were pigmented individuals. It seems unlikely that 
pigmentation of itself was responsible for the resistance, although 
it may well have been associated with the true cause. Uchida, 1431 
who earlier had isolated pneumococci from guinea pigs suffering 
from spontaneous infection, noted irregularities in the results fol- 
lowing the subsequent subcutaneous, intraperitoneal, and intraven- 
ous injection of the strains into normal guinea pigs. The author 
assumed that the discrepancies in the outcome of the experiments 
were due to varying degrees of resistance possessed by the indi- 
vidual animals, but offered no specific explanation for the differ- 
ences. 

Wamoscher (1927) 1479 observed that scurvy and chronic tuber- 
culosis in the guinea pig were diatheses favoring spontaneous pneu- 
mococcal infection. These and similar debilitating conditions may 
likewise lower resistance to experimental infection. Schmidt-Wey- 
land and Koltzsch* found that scorbutic guinea pigs could easily 
be infected when pneumococci were introduced into the body by in- 
halation or feeding. 

THE MOUSE 

The white races of the mouse family, because of low cost, ease of 
handling, great susceptibility, and general uniformity of reaction, 

* Quoted by Neufeld and Schnitzer. 



188 BIOLOGY OF PNEUMOCOCCUS 

are ordinarily chosen for the isolation of pneumococci, the pre- 
liminary testing of antigenic substances, and for the determination 
of type-specificity and potency of diagnostic and curative immune 
serums. Albino strains are preferable to strains of pigmented or 
wild varieties because of their lower resistance to infection. Pure- 
line races, with their inherited uniformity of susceptibility, would 
be ideal types for routine investigative purposes were it not for 
their present scarcity and prohibitive cost. That susceptibility or 
resistance to infection are transmissible characters and that the 
breeding of races with either high or low degrees of susceptibility 
can be accomplished by proper selection has been demonstrated by 
Irwin and Hughes 670 in the case of the rat for bacteria of the en- 
teric group. 

The extreme susceptibility of the mouse to subcutaneous, intra- 
peritoneal, or intravenous injection of Pneumococcus is shown by 
the rapid invasion of the blood stream and the death of the ani- 
mals without localization of the infection. Introduced by any of 
these routes Pneumococcus may be highly infective for mice. When 
the virulence of a strain of Pneumococcus has been exalted by suc- 
cessive mouse passage, the cultures used for intraperitoneal injec- 
tion into a mouse may be so diluted that, although the amount used 
for inoculation may yield only one or even no colonies, infection 
will frequently follow. In fact, Wamoscher 1478 has demonstrated by 
micromechanical isolation of single cells that one pneumococcus 
may suffice to infect a mouse. It is for this reason, therefore, that 
the mouse is so admirably adapted for the detection of pneumo- 
cocci in infective material, for determining virulence, for testing 
the immunizing action of antigens, and for measuring the protec- 
tive power of specific immune serum. 

An observation concerning the dominance of one type of Pneu- 
mococcus in causing general infection in the mouse following the 
injection of an inoculum containing pneumococci of several types 
was described by Etinger-Tulczynska. 368 When mixtures of equal 
parts of cocci of different types were administered either by the 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 189 

subcutaneous, intraperitoneal, or intravenous route or by all three 
routes simultaneously, the organisms of one type would gain the 
ascendancy and suppress the pneumococci of the other type even 
at the site of inoculation. 

Webster and Clow 1494 found individual differences in mice to in- 
tranasal infection with Pneumococcus. Some mice were completely 
refractory, some became carriers, and others developed various 
forms of infection ranging from lobar pneumonia to septicemia. 
Animals which showed high resistance to a strain introduced 
through the nose might exhibit moderate or high susceptibility to 
the same strain injected intraperitoneally. Whether the grades of 
infectivity were due to differences in the virulence of the organisms 
or to degrees of susceptibility of the host was not ascertained. 

Distinct differences in the reactivity of mice of diverse races to 
pneumococcal infection has been demonstrated by Rake. 1117 " 8 In 
general, reactivity was found to be influenced by the type of or- 
ganism used for inoculation as well as by innate characters pe- 
culiar to the breed of the animal. For example, a single type of 
Pneumococcus produced in mice of the same breed lesions which 
were similar and predictable. Lesions differing quantitatively could 
be produced in various breeds of mice by inoculation of the same 
type of organism, but inoculation of cultures of the various types 
into mice of a single race produced lesions differing in quality. Ex- 
periments in which mice were infected intranasally and intrave- 
nously revealed that lesions in the lung and other organs varied 
with both the type of culture and the strain of mice used. 

The constancy of the results obtained in infection experiments 
with this animal species is further conditioned by the age of the 
individuals employed. Moreover, there is greater regularity in the 
behavior of mice of approximately sixteen to twenty-one grams in 
weight to artificial infection, and this fact is especially significant 
when mice are employed for testing the potency of antipneumococ- 
cic serum. More recently, Goodner and Miller (1935), 540 extending 
Goodner's work on the rabbit, investigated the physiological vari- 



190 BIOLOGY OF PNEUMOCOCCUS 

ables responsible for the resistance of the individual mouse to pneu- 
mococcal infection by studying the capacity of animals of a sin- 
gle strain to utilize the protective properties of antipneumococcic 
serum. The authors concluded that the important variables were 
body-weight and the number of cells in the peritoneal cavity fol- 
lowing the intraperitoneal injection of pneumococci and homolo- 
gous immune serum, which in turn resolved itself into the number 
of monocytes present. The dominant factor in determining sus- 
ceptibility, other factors being equal, was the relation between the 
number of monocytes and the number of pneumococci in the peri- 
toneal cavity at the time of the injection of culture and serum. 

The resistance of mice to infection by inhalation may be de- 
creased by the previous administration of alcohol. Stillman and 
Branch (1924, 1930, 1931 ) 1337 ' 13401 found that inspired pneumo- 
cocci rapidly disappeared from the lung of normal mice and rarely 
caused septicemia, but in alcoholized mice the organisms persisted 
in the lung for a longer period and fatal septicemia was frequent, 
while pulmonary localization of the infection occurred in mice pre- 
viously immunized either actively with heat-killed pneumococci or 
passively with homologous immune serum. The observations of 
Branch and Stillman 148 were confirmed by Lange and Keschis- 
chian, 785 who succeeded, but only with difficulty, in inducing pul- 
monary infection in mice through inhalation. The latter authors 
also found mice to be resistant to percutaneous and peroral inocu- 
lation with pneumococci. 

The frequency with which colonies of white mice are infected 
with the so-called mouse typhoid due to Bacillus typhi murium is a 
factor to be considered when selecting mice for experimental pur- 
poses. The disease, so often remaining latent, may not be evident, 
but it may nevertheless greatly alter the reaction of the animals to 
experimental inoculation. 

THE RAT 

The rat is a near zoological relation of the mouse and is highly 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 191 

receptive to pneumococcal infection, but it is seldom used in re- 
searches on Pneumococcus. Lamar* and also Neufeld and Haen- 
del* proved the great susceptibility of the rat to pneumococcal in- 
fection. The subcutaneous injection of 10" 5 cubic centimeters of a 
virulent strain caused septicemia which resulted two days later in 
death of the animal. 

McDowell 878 studied the effect of high air temperatures, com- 
bined with different degrees of humidity, on resistance of rats to 
intraperitoneal inoculation of pneumococci. It appeared that after 
an exposure for two weeks to a temperature of 83°F., with the hu- 
midity varying from 44 to 72 per cent, rats exhibited greater re- 
sistance than did rats kept at medium temperatures. High or low 
humidity with temperatures between 65° and 72°F. were unfavor- 
able to the survival of the test animals. However, when rats accus- 
tomed to moderate temperatures (67° to 71°F.) were inoculated 
intraperitoneally with Pneumococcus and then exposed to a higher 
temperature (83°F.) there was a lowering of resistance. 

Rats, as well as mice, proved to be susceptible to pneumococcal 
infection when the organisms in the form of pulverized, dried, in- 
fected blood or spleens were insufflated into the trachea by Kramar 
and Gyiire. 757 McDaniels 876 " 7 employed rats in an investigation of 
the immunizing action of orally administered pneumococcal vac- 
cines and found the species to be suitable for the purpose. 

THE MONKEY 

Monkeys in captivity are susceptible to spontaneous pneumo- 
coccal disease. Blake and Cecil (1920) 128 reported that pneumonia 
might occur in epidemic form, due to spread of infection from ani- 
mal to animal, when conditions favoring close contact existed. The 
disease in monkeys, according to the authors, was identical in its 
clinical features, complications, and pathology, with lobar pneu- 
monia experimentally produced in monkeys by the intratracheal 
injection of Pneumococcus and with lobar pneumonia in man. Two 

* Quoted by Neufeld and Schnitzer. 



192 BIOLOGY OF PNEUMOCOCCUS 

strains of the organism isolated from the sick monkeys were pneu- 
mococci of Group IV. At that time, the authors 126 " 7 demonstrated 
that lobar pneumonia could be consistently produced in members 
of this animal species (Macacus syrichtus and Cebus capucinus) 
by the intratracheal injection of minute amounts of culture of a 
virulent Type I strain. When large quantities of culture were in- 
troduced into the nose or throat, no lobar pneumonia developed, 
but the animals became carriers of the inoculated organism and re- 
mained so for a period of at least a month. 

The susceptibility of the monkey (Macacus rhesus and Cero- 
pithecus callitrichus) to intracranial or intraspinal inoculation 
with Pneumococcus was demonstrated in 1912 by Lamar, 775 who 
described the meningitis following the injections. The experimental 
disease resembled pneumococcal meningitis in man but ran a more 
rapid course and was invariably fatal. 

In studying pneumococcal infection and immunity in monkeys, 
Cecil and Steffen 211 found that Macacus rhesus was most resistant 
to Type I Pneumococcus and rarely developed true lobar pneu- 
monia ; Cebus capucinus occupied an intermediate position, occa- 
sionally showing typical lobar infection but more often interstitial 
or patchy lesions ; while the Philippine monkey, Macacus syrich- 
tus, was the most susceptible and was preferable for inoculation 
experiments because animals of that species develop true lobar 
pneumonia. 

The existence of the carrier state in normal stock monkeys was 
also shown by School and Sellards, 1246 who recovered an avirulent 
strain of Pneumococcus from the Philippine monkey, Pithecus 
philippinensis. The authors also succeeded in inducing pneumonia 
in the animals by intratracheal inoculation with a small dose of a 
broth culture of Type I Pneumococcus that was highly virulent 
for mice. 

Blake and Cecil, 126 using strains of Type I, II, III, and Group 
IV pneumococci, by intravenous injection failed to produce pneu- 
monia in monkeys of the Macacus syrichtus species but reported 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 193 

that a fatal septicemia followed inoculation by that route. No 
greater success followed the attempts of School and Sellards, al- 
though the latter authors likewise observed fatal systemic infec- 
tion after intravenous injection of a Type I culture. Cecil and 
Steffen 214 " 5 were able by injecting cultures of Types I, II, and III, 
and of one Group IV strain into the trachea to incite pneumonia 
in Macacus rhesus, Macacus syrichtus, and Cebus capucinus. 

Among other references to the experimental production of lobar 
pneumonia in monkeys by the intratracheal implantation of viru- 
lent pneumococci may be mentioned the report of Francis and Ter- 
rell, 476 who were able to induce pneumonic disease in Cynomolgos 
monkeys with a Type III strain. The authors reported that the 
type of infection following small doses given in winter was similar 
to the type resulting from large doses administered in warm 
months. The authors also noted marked individual variations in 
the monkeys used. According to Stuppy, Falk, and Jacobson, 1853 
Macacus rhesus and Cebus capucinus were highly resistant to in- 
tratracheal inoculation of virulent Type I Pneumococcus. None of 
the thirteen animals injected developed lobar pneumonia. Three 
monkeys died from pneumococcal infection but the lungs appeared 
to be normal, except for an increase in the number of polymor- 
phonuclear leucocytes in the interstitial tissue, blood vessels, and 
bronchi. 

THE CAT 

Feline animals are unsuitable for studies on pneumococcal infec- 
tion. Robertson, Woo, Cheer, and King 1152 are apparently the only 
authors to have reported experiments on cats in which it was pos- 
sible by intrapleural injection of cultures of Type I and II pneu- 
mococci to produce lobar pneumonia. However, only one animal of 
those inoculated developed the disease. 

THE DOG 

The first reference to the use of dogs in research on Pneumococ- 



194 BIOLOGY OF PNEUMOCOCCUS 

cus is probably that of Friedlander (1883), 487 who injected aque- 
ous suspensions of gelatin cultures of cocci isolated from pneu- 
monia patients into the lungs of four dogs, only one of which suc- 
cumbed. The animal at necropsy showed red and gray hepatization 
of the lung, and from the areas Friedlander succeeded in recover- 
ing typical encapsulated diplococci. In the same year, Talamon 1378 
failed to infect dogs with a mixed culture containing lanceolate 
cocci grown from the exudate of a pneumonic lung. The refrac- 
toriness of dogs was also demonstrated by Monti, 905 " 7 who reported 
that no reaction attended subcutaneous injection. However, he 
succeeded in producing meningitis in the dog after subdural inocu- 
lation with pneumococci. 

Salvioli (1884) 1215 was more successful in infecting dogs with 
encapsulated cocci obtained from the pleural and pericardial exu- 
dates of pneumonia patients, but the animals so treated failed to 
develop the typical lesions of pneumonia. In 1912, Lamar and 
Meltzer, 772 by means of a catheter introduced through the larynx 
and bronchus, implanted pneumococci in the lungs of dogs. Of 
forty-eight test animals, forty-two developed lobar pneumonia 
with a fatality rate of 16 per cent. Wadsworth 1457 employed the 
method to study phagocytosis in similarly infected animals. In the 
next year, Wollstein and Meltzer 1539 " 40 in two communications de- 
scribed the results following the insufflation of avirulent and of 
heat-killed pneumococci into the lungs of this domestic animal. The 
injection caused pulmonic congestion with exudate but, in general, 
the framework of the lung was unaffected and the process was non- 
fatal. 

Similar results were reported by Newburg, Means and Porter 
(1916), 1004 by Kline (1917), 726 Wadsworth (1918), 1459 Leake, 
Vickers and Brown (1924), 793 Christie, Ehrich and Binger 
(1928), 233 and by Coryllos (1929), 279 all of whom employed the 
same technique of using the bronchoscope for the injection of 
Type I culture as used by Henderson, Haggard, Coryllos, and 
Birnbaum (1930) 636 with a culture of Type II Pneumococcus. 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 195 

With strains of both Types I and II introduced into a terminal 
bronchus by means of a catheter, Terrell, Robertson, and Cogge- 
shall (1933) 1387 made fluoroscopic studies of the lobar pneumonic 
process which ensued in every instance when appropriate doses of 
culture were administered. In the experiments of the last-named 
authors the course of the pneumonia was short, averaging four or 
five days, while recovery, which took place in the majority of the 
animals so infected, was abrupt and simulated the crisis occurring 
in man. Similar experiments with pneumococci of Types I and III 
Mere described in 1934 by Lieberman and Leopold. 813 The article 
by Terrell, Robertson, and Coggeshall 1387 contains excellent de- 
scriptions of the pathological processes observed following intra- 
bronchial injection of pneumococci in dogs. 

Bull (1916) 173 injected dogs intravenously with pneumococci. 
Invasion of the blood stream appeared twenty-four to forty-eight 
hours after the injection. The septicemia reached a climax in 
four to five days, then abruptly declined, the blood becoming sterile 
in one to three days after the peak of the septicemia was reached. 
In some of the animals so injected meningitis occurred. 

The results of a study of meningeal inoculation in dogs have 
been described by Stewart. 1323 The injection of Type I and Type 
II organisms into the ventricle, the cistern, and the lumbar region 
was followed by purulent meningitis with accompanying bacteri- 
emia when the cultures used were of high virulence. Not all the ani- 
mals infected succumbed, but those that died showed pathological 
changes not entirely comparable to those found in pneumococcal 
meningitis in man. 

THE HORSE 

The susceptibility of horses to infection with Pneumococcus, 
mentioned by Neufeld and Schnitzer, is a fact all too familiar to 
those engaged in the manufacture of therapeutic antipneumo- 
coccic serum. The possession of a high degree of specific, active 
immunity to pneumococci of a given serological type may fail to 



196 BIOLOGY OF PNEUMOCOCCUS 

protect the horse against the subcutaneous or intravenous injec- 
tion of living, virulent strains of the same type. Abscesses, pneu- 
mococcemia, endocarditis, and pneumonitis, sometimes culminating 
in fatal pneumothorax, may follow immunizing injections of living 
cultures, even when the serum of the animal under active immuniza- 
tion contains specific agglutinins and protective antibodies in suf- 
ficient quantity to qualify it for therapeutic use. This paradoxical 
phenomenon will be discussed in a later chapter. 

BIRDS 

Hens and doves have been found to be refractory to infection 
with Pneumococcus by Fraenkel,* Gamaleia, 498 and by Kyes, 766 
among others. Kindborg (1905) 713 apparently was the only worker 
who claimed to have succeeded in demonstrating pathogenicity of 
Pneumococcus for pigeons. Kyes studied the cellular reaction in 
tissues of pigeons injected intraperitoneally with virulent pneumo- 
cocci, and reported that the invading organisms were rapidly 
withdrawn from the blood stream and localized in the liver and 
spleen. Because the ultimate localization of the cocci in both of 
these organs was within a type of fixed phagocyte — the hemo- 
phage — common to both organs, Kyes concluded that the phago- 
cytic destruction of pneumococci by hemophages is so extensive 
and so rapid as actually to constitute an important, if not indeed 
the determining, factor in the resistance of birds to pneumococcal 
infection. The natural resistance of hens and doves may, however, 
be lowered to the point of susceptibility through vitamin deficiency 
or by the administration of poisons, as reported by Strouse, by 
Guerrini, and by Corda.* 

Virulence of the Organism 

In any discussion of the pathogenicity of an organism, it should 
be borne in mind that virulence is a purely relative term. The fac- 
tors which enter into the determination of the invasiveness of a 

* Quoted by Neufeld and Schnitzer. 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 197 

given strain of Pneumococcus are the serological type; the vital 
condition of the coccus at the time of trial as shown by the posses- 
sion of a capsule; the mass of culture injected; the site chosen for 
inoculation ; the species, and even the variety and the individual 
idiosyncrasies of the animals selected. There also enters the ques- 
tion whether the malignancy of a culture is due to a uniform viru- 
lence of all the cocci in a culture or to the presence of a few or- 
ganisms of especially high virulence accompanied by other organ- 
isms possessing less or no invasive power. 

Pneumococci of the various serological types as they exist in 
the lesions, exudates, or secretions of infected animals, or when 
freshly isolated from these sources, are pathogenic for other ani- 
mals of the same species and, depending upon the serological type, 
may be virulent for animals of a different species. When a strain 
is passed serially through the bodies of animals of a given species 
it may acquire an elevated infectivity for other members of that 
species. Virulence may reach a permanent zenith or may decline 
but, virulent though an organism may be for the species used, it 
by no means follows that it is correspondingly pathogenic for 
animals of a different species. 

During propagation outside the body the conditions of cultiva- 
tion effect profound changes in the integrity of Pneumococcus, 
which, in turn, affect its pathogenic powers. Conditions that serve 
to maintain the organism in the highest state of metabolic activity 
with maximal production of capsular substance favor increase and 
maintenance of virulence. Conditions that, on the contrary, induce 
dissociation and degradation of the organism, by being inimical 
to capsule formation, lessen or destroy virulence. 

FRESHLY ISOLATED STRAINS 

In considering some of the earlier reports dealing with the 
pathogenicity of strains isolated from cases of lobar pneumonia 
and other pneumococcal infections, it should be remembered that 
many of the studies were conducted at a time before pneumococci 



198 BIOLOGY OF PNEUMOCOCCUS 

were divided into serological types. Eyre and Washbourn 378 in 
1899 gave descriptions of four strains of pneumococci, of which 
three, isolated from infections in man, were considered by the au- 
thors to be parasites, and one strain, from the normal human 
mouth, was looked upon as a saprophyte. The first three strains 
displayed great capabilities both for acquiring and for retaining 
a high degree of virulence, whereas the fourth culture possessed a 
low capacity in both respects. 

The severity or lack of severity of an infection may depend on 
a preceding or accompanying infection with another bacterial spe- 
cies. For example, Sinigar (1903) 1292 described the ascending viru- 
lence of a respiratory infection among the staff and patients in the 
Leavesden Asylum. Beginning as a brief, indefinite illness, the dis- 
ease gradually increased in severity, occasionally showing bron- 
chial symptoms, and then developed into lobar pneumonia with a 
high fatality-rate. In all the cases, it was alleged that pneumococci 
were present in large numbers but, since there is no reference in 
the text to cultivation or virulence tests, it is impossible to say 
whether the same strain of Pneumococcus — if the organism was a 
pneumococcus — gradually gaining virulence, was responsible for 
the epidemic, or whether pneumococci of relatively low virulence 
were succeeded by a type having greater infectivity. 

Kindborg (1905), 713 in a study of a large number of strains ob- 
tained from normal and pneumonic sputum, empyema pus, and 
other sources due to pneumococcal infection, observed wide limits 
in the pathogenicity of the different cultures. The organisms from 
cases of pneumonia were said usually to be the most virulent, while 
strains isolated from local inflammatory processes were generally 
avirulent. Whittle 1519 determined the virulence for mice of sixty 
strains of pneumococci collected at random from infections in man, 
and concluded from his tests that pneumococci by virtue of their 
pathogenic powers could be divided into at least two groups ; those 
of high virulence were responsible for such well-recognized clinical 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 199 

entities as lobar pneumonia and bronchopneumonia, and those of 
low virulence were associated with minor illnesses or with disease 
occurring in persons already debilitated. Whittle denied the exist- 
ence of any relation between serological type and pathogenicity. 

Gundel and Wasu (1931) 578 reported that the virulence of a 
given type of Pneumococcus reached a maximum at the height of 
the disease process and that in the complications of lobar pneu- 
monia in man, such as meningitis and otitis media, only strains of 
the highest virulence were found, the organisms usually belonging 
to the fixed serological types. Further discussion of the infectious- 
ness for man of pneumococci of the various serological types will 
be found in the following chapter. 

NUMBERS OF COCCI REQUIRED TO INFECT 

Among reports on the quantitative determination of the infec- 
tive ability of pneumococcal strains there may again be mentioned 
the observations of Wamoscher (1926). 1478 L T sing a strain of Type 
III Pneumococcus, counting the number of cocci in the inoculum 
by means of the Peterfi micromanipulator, and injecting the or- 
ganisms subcutaneously into white mice, the author found that ap- 
proximately one-quarter of the number of mice so treated suc- 
cumbed to pneumococcal infection in two to four days after re- 
ceiving an inoculation of one pneumococcus. The percentage of 
fatal infections rose with an increase in the number of organisms 
injected. Using only freshly isolated strains of pneumococci grown 
in broth from the culture obtained from a single mouse passage 
after isolation from a human source, and injecting the culture in- 
traperitoneally into white mice, Gundel and Wasu noted marked 
differences in the virulence of strains within a type, although, as a 
rule, representatives of Types III, II, I, and IV possessed degrees 
of virulence in the order named, the average minimal lethal dilution 
for all Type III strains being 1 to 100,000,000, for Type II cul- 
tures 1 to 10,000,000, for Type I cultures 1 to 1,000,000, while 



200 BIOLOGY OF PNEUMOCOCCUS 

for pneumococci of Type IV the killing dose varied from 1 to 100 
to 1 to 1,000. One of the most virulent strains tested, a Type IV 
culture, was fatal in a dose of 10 8 cubic centimeters. 

Petrie and Morgan 1085 in the same year reported the results ob- 
tained in an investigation of the factors influencing the lethal 
power for mice of a virulent culture of Type I Pneumococcus. 
Stocks of mice from different sources appeared to show some va- 
riation in susceptibility, and a small proportion — from 5 to 10 
per cent — exhibited an innate resistance to small doses of pneu- 
mococci. The weight factor of the test animals failed to affect the 
mortality, although it modified the survival time. However, the au- 
thors decided that the density and virulence of the culture deter- 
mined its lethal power, and that this power could be calculated 
when a reasonable number of mice were used. The criteria, by 
means of which the minimal fatal dose of any culture of Type I 
Pneumococcus could be specified, were the percentage fatality, the 
mean death-time, and the distribution of deaths at appropriate 
time intervals in a group of mice receiving a definite dose of the 
test culture. 

AVENUE OF INOCULATION 

In general, the virulence of a given organism varies with the 
tissue into which it is introduced. Direct inoculation into the blood 
stream is the most rapidly and most surely lethal route, followed 
in order of effectiveness by injection into the anterior chamber of 
the eye, into the peritoneum, under the skin, into the skin, into the 
lung either by direct puncture or through the bronchi, and lastly 
by way of the mouth. 

DETERMINATION OF VIRULENCE 

While the intraperitoneal injection of white mice is the pre- 
ferred method for testing the virulence of pneumococcal cultures, 
other methods have been described. The rabbit, because of cost and 
individual and special idiosyncrasies, is less valuable. The relation 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 201 

between virulence and electrophoretic potential of different strains 
and serological types of Pneumococcus has been studied by Falk 
and his associates, Gussin and Jacobson. 379 According to the au- 
thors the highest potentials were uniformly found for Type III 
organisms, while the most probable sequence of decreasing poten- 
tials was from Type III to I to II and then to Group IV strains. 
The order corresponded to the decreasing sequence of virulence of 
the cultures for white mice. Washing the cultures increased elec- 
trophoretic velocities, but the order remained the same. From the 
results obtained with stock cultures, the authors anticipated that 
strains isolated from fatal cases of pneumonia might show higher 
potentials than would strains of the same type isolated from non- 
fatal cases. The experimental data confirmed the assumption for 
Types II and III, and Group IV, but contradicted it for Type I 
strains. Falk, Gussin, and Jacobson 379 concluded that electropho- 
retic potential was related in some fundamental manner to viru- 
lence "as well as to phagocytability, agglutinability, capsule for- 
mation, and other characters of microorganisms. In a later com- 
munication, Jacobson and Falk, 675 after a study of this electrical 
phenomenon in pneumococcal variants, reported that in all cases 
studied alterations in virulence were accompanied by parallel al- 
terations in electrophoretic potential and by reciprocal alterations 
in agglutinability. 

The results obtained by Thompson (1931 ) 1396 in some respects 
contradict those reported by Falk and his colleagues. While the 
electrophoretic migration of representatives of the first three 
pneumococcal types was observed to decrease in the order III, I, 
II, as previously noted by Falk, Gussin, and Jacobson, exaltation 
or degradation of virulence respectively by mouse passage and by 
growth in increasing concentrations of ox bile were unaccompa- 
nied by any constant differences in the original velocity of migra- 
tion. 

Welikanow and Michailowa (1930) 1513 claimed that variation 
in the ability of pneumococci to ferment glucose is an indicator 



202 BIOLOGY OF PNEUMOCOCCUS 

of the virulence of the strain, but the evidence presented is too 
meager to be convincing. 

SUBSTANCES THAT ENHANCE VIRULENCE 

In Chapter III mention was made of substances or preparations 
which possessed the ability to increase the invasive power of pneu- 
mococci. The virulin of Pittman and Falk, 1092 the extracts of Pitt- 
man and Southwick, 1093 the leucocidin of Oram, 1032 and the toxic 
autolysate of Parker and Pappenheimer 1063 are examples of sub- 
stances which to a greater or less degree possess this property. A 
similar action was observed by Sia, 1266 who found that the addi- 
tion of a very small amount of soluble specific substance or of 
young broth cultures of pneumococci to avirulent cultures of 
homologous type when mixed with rabbit or cat serum-leucocyte 
mixtures favored the growth of the organism. The authors inter- 
preted the result as indicating that soluble specific substance had 
the power of rendering virulent an avirulent Pneumococcus of the 
same serological type. Sia and Zia 1275 reported that the injection 
of Type II soluble specific substance into rabbits depressed the 
resistance to such an extent that the animals succumbed to the 
intravenous injection of an otherwise sublethal dose of pneumo- 
cocci of the same serological type. The authors left undecided the 
question whether the result was to be ascribed to a heightened 
susceptibility of the animal or to an enhanced virulence of the or- 
ganism. 

A somewhat similar effect was obtained by Nungester, Wolf, and 
Jourdonais, 1020 who added gastric mucin to twenty-four-hour 
broth cultures of Type II pneumococci and injected the mixture 
intraperitoneally into mice. Control mice received similar injec- 
tions of the culture suspended in saline instead of mucin solution. 
At only one dose level was any marked difference noted in the per- 
centage of survivals among the test and the control animals. The 
effect was not apparent when the injections were made intra- 
venously or subcutaneously. Any action of mucin, in spite of its 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 203 

correspondence in chemical structure to soluble specific substance 
of Pneumococcus, must have been non-specific, and this surmise is 
strengthened by the fact that a slight adjuvant action in favoring 
infectivity was also noted when streptococci instead of pneumo- 
cocci were the organisms so treated and inoculated. 

The influence of pneumococcal autolysates on the invasiveness 
of pneumococci injected intradermally into rabbits was studied by 
Goodner. Whereas autolysates favored the pathogenicity of the 
culture used, they failed to alter its virulence. The evidence, taken 
as a whole, favors the hypothesis that the action of culture fil- 
trates, extracts, autolysates, or specific capsular polysaccharides 
in increasing the infective power of pneumococci is to interfere 
with the natural defensive processes of the body and thereby lower 
the host's resistance rather than to add to the virulence of the cell 
itself. 

CULTURAL CONDITIONS AND VDIULENCE 

Influences that stimulate the anabolic processes of the pneumo- 
coccal cell make for virulence. Enrichment of the cultural medium 
with blood, normal serum, or other growth accessories, the proper 
concentration of hydrogen ions, and optimal temperature of in- 
cubation enable the organism to elaborate capsular substance, 
which, as will be shown in other portions of the present volume, 
may determine the invasive power of a given strain. Frequent 
transplantation in favorable media of pneumococcal cultures taken 
at the period of maximal growth not only maintains but increases 
virulence (Wadsworth and Kirkbride 1471 ). Repeated transfer by 
means of the automatic device of Felton and Dougherty 423 of 
young cultures of a pure-line strain of Type I Pneumococcus into 
a fresh supply of sterile skimmed milk caused an avirulent strain 
to acquire a virulence ten million times greater than that of the 
original culture. Within a wide range, the hydrogen ion concen- 
tration of the milk appeared to have only a slight effect on viru- 
lence when transplants were made at two-hour intervals. However, 



204 BIOLOGY OF PNEUMOCOCCUS 

when the reaction of the medium was adjusted to pH 8 or 9, viru- 
lence decreased ; the more alkaline the reaction the more rapid the 
decrease. 

When cultivated in broth by the same method, 425 the Type I cul- 
ture lost virulence as the reaction of the medium by adjustment 
became more acid. Virulence fluctuated with the frequency of trans- 
fer of the organism to fresh broth. In the case of transplants made 
every hour, virulence immediately decreased. When the interval 
was two, four, or eight hours, there was first a rise and then a fall 
of virulence, the rise being greatest in the case of eight-hour and 
least with the two-hour transfers. With the hydrogen ion concen- 
tration at set points the amount of meat infusion influenced the 
virulence of the culture, the unfavorable action varying in inverse 
proportion to the concentration of meat in the substrate. The ad- 
dition of glucose neutralized the action of the meat. Peptone in 
the broth also affected the virulence of the strain studied, Felton 
and Dougherty reporting that the nutrient in a concentration of 
2 per cent maintained and even increased virulence of the strains 
studied. 

In a later communication (1932), Felton 411 " 2 described other 
nutritional factors that affect the virulence of pneumococci. Media 
made from calf lung or heart or from the skeletal muscles of the 
horse maintained for a long period of time the virulence of the cul- 
tures used ; conversely, media prepared from calf spleen led to a 
decrease in pathogenicity. Normal horse serum, or specific immune 
serum freed from protective antibody, preserved virulence. Media 
made from rabbit muscle were less suitable for the purpose than 
media prepared in the same way from the meat of guinea pigs. 
When grown in the automatic transfer device in a medium which 
aerobically maintained the virulence of a Type I culture, the addi- 
tion of pure oxygen or pure carbon dioxide lowered virulence, but 
no change in infectivity was noted when the organisms were culti- 
vated in the presence of nitrogen. Gradual increase in the tem- 
perature of incubation from 36.5° to 42° over a period of ten 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 205 

days, in a sample of medium otherwise suitable for maintenance of 
virulence, resulted in a decrease in infective power of the pneumo- 
cocci studied. 

According to Gaskell, 499 a medium made from beaten eggs was 
satisfactory for preserving the virulence of pneumococcal cultures, 
but the interpolation of fortnightly passages through the mouse 
was necessary in order to restore waning infectivity. For addi- 
tional methods for assuring or preserving the virulence of cul- 
tures of Pneumococcus or of fluids or tissues containing the or- 
ganism, the reader is referred to Chapter II. 

STRAIN VARIATIONS IN VIRULENCE 

The ability of a given strain of Pneumococcus to retain its viru- 
lence over a long period and then suddenly to lose its invasive 
power is too well known to require extended comment. Browning 
and Gulbransen (1923) 161 described a culture of Type I Pneumo- 
coccus, passed more than ninety times through mice during a pe- 
riod of six years, which, when preserved in the dried spleen of an 
infected mouse, exhibited marked variations in its ability to infect 
mice. The conclusions reached by Gaskell (1928) 501 in a study of 
the pathogenicity of single strains of pneumococci of Types I, II, 
and III and Group IV may be cited. The virulence of Type II 
strains was less than that of Type I organisms for both mice and 
rabbits ; the pathogenicity of members of Type III was lower than 
that of Type I for rabbits, mice, and also man ; whereas the viru- 
lence of the Group IV cultures obtained from severe infections in 
man was, if anything, higher than that of Type I pneumococci. 

Other individual strain differences with respect to the ability of 
pneumococci to infect experimental animals have been reported by 
Webster and Clow (1933). 1494 The degree of virulence of a strain 
when inoculated into the nose of the mouse failed to parallel intra- 
peritoneal virulence in 50 per cent of the strains studied — high 
intranasal invasiveness being accompanied by either high or mod- 
erate intraperitoneal virulence, and low intranasal by high, mod- 



206 BIOLOGY OF PNEUMOCOCCUS 

erate, or low intraperitoneal virulence. Type III strains were of 
relatively high intranasal and intraperitoneal infectivity ; Type II 
organisms were low in intranasal but high or moderate in intra- 
peritoneal virulence ; Type I strains were all low in intranasal but 
either high or moderate in intraperitoneal virulence ; while the ma- 
jority of strains of other types were low both in intranasal and 
intraperitoneal pathogenicity. Intranasal virulence of pneumo- 
cocci was not enhanced by animal passage, but nasal passage re- 
duced the intranasal virulence to zero without altering intraperi- 
toneal virulence, colony form, or agglutinative specificity of the 
strains. Passage by the intraperitoneal method maintained the 
characteristic level of intranasal virulence for a period, increased 
intraperitoneal virulence in some instances, but did not affect 
colony form or agglutinative properties. 

VIRULENCE IN RESPECT TO ANIMAL SPECIES 

In addition to individual differences in pneumococci, variations 
in the susceptibility of various animals are factors to be reckoned 
with in the evaluation of the virulence of a given type or strain. 
An organism may possess superior virulence for animals of one 
species and yet fail to infect those of another species, although the 
animals may readily be infected with representatives of a different 
variety or type. Eyre and Washbourn, 374 in 1898, reported that, 
after increasing the virulence of a culture for guinea pigs by re- 
peated passage through a series of these animals, there was no 
change in the virulence of the organism for mice. Fourteen years 
later, Neufeld and Ungermann 1001 " 2 observed that while repeated 
passage of a strain of Pneumococcus through the guinea pig ele- 
vated the virulence of the organism for animals of the same spe- 
cies, similar serial propagation in mice failed to increase the in- 
fectiousness of the strain for the guinea pig. 

In 1912, Truche and Cotoni 1423 reported that strains of pneumo- 
cocci isolated from human sources were rarely virulent for rabbits 
or guinea pigs. Strains virulent for rabbits were always infective 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 207 

for mice, while pneumococci that were invasive for guinea pigs 
possessed high virulence for both rabbits and mice. In a second 
paper, Truche and Cotoni 1424 described the effect of passing four 
strains of Pneumococcus through mice, rabbits, and guinea pigs. 
When strains virulent for mice were serially propagated through 
animals of that species the organisms maintained their high viru- 
lence but acquired no greater virulence over the original culture 
for rabbits or guinea pigs. The same condition held true when 
pneumococci were passed through rabbits, but there was no altera- 
tion in the infectiousness of the strains for guinea pigs or mice. 
Cultures subjected to passage through guinea pigs showed no in- 
crease in pathogenicity for animals of that species or for mice or 
rabbits. 

Twenty strains of Type III Pneumococcus studied by Levy- 
Bruhl (1927) 804 were highly virulent for guinea pigs, moderately 
so for mice, and very feebly or not at all virulent for rabbits. The 
failure of many strains of Type III Pneumococcus to infect rab- 
bits was also reported in the same year by Tillett. 1403 Of eleven 
strains isolated from human sources, ten possessed low virulence 
for rabbits despite the fact that all the strains possessed large 
capsules and a high degree of virulence for mice. The odd strain 
was rendered highly infectious for rabbits, and since it possessed 
no other biological property demonstrably different from the other 
ten strains, Tillett concluded that its individual virulence must re- 
side in some additional property. 

The lack of virulence for rabbits of the majority of Type III 
pneumococci has perplexed many bacteriologists, and no explana- 
tion has been forthcoming for the disparity in virulence of Type III 
strains until the recent communication of Enders and Shaffer. 860 
Evidence was obtained that a correlation exists between the in- 
ability of a strain to grow at 41° and virulence, since only among 
the thermoresistant strains were found those possessing the prop- 
erty of rabbit virulence. The attribute is constantly but not ex- 
clusively associated with all Type III pneumococci, and was con- 



208 BIOLOGY OF PNEUMOCOCCUS 

sidered by the authors as a prerequisite, but not the sole factor, 
in determining the virulence of an organism for rabbits. Continu- 
ing the investigation, Shaffer with Enders and Wu 1259 studied 
two smooth strains of the same organism, one virulent and the 
other relatively avirulent for rabbits. Although no antigenic differ- 
ences between the two organisms were revealed by cross-absorp- 
tion of agglutinins from homologous antiserum, the latter strain 
lost its capsule in dextrose serum-broth cultures about eight hours 
earlier than was the case with the rabbit-virulent organism. With 
the loss of capsule there was marked shrinkage in volume, altera- 
tion in the zone of acid agglutination, susceptibility to agglutina- 
tion in antirough pneumococcic serum and to phagocytosis. 

The results which followed the intravenous injection into rabbits 
of the two strains varied with the state of the capsule. A culture 
of either strain became susceptible to the blood-clearing mecha- 
nisms contemporaneously with the onset of capsular degeneration 
and the beginning of other concomitant changes at the surface of 
the organism which occurred much earlier with the less virulent 
strain. Phagocytosis by the leucocytes of the normal animal either 
in vitro or in vivo was observed only at such a time as the capsule 
had become impaired. The authors (Enders, Shaffer, and Wu) 361 
concluded that virulence for rabbits of the two strains of Type III 
Pneumococcus does not imply that this animal possesses a defen- 
sive mechanism which is absent in other species, since it was pos- 
sible to demonstrate similar differences in virulence when the or- 
ganisms were injected intravenously into mice. Therefore, "the 
factors determining the degree of virulence of these strains are to 
be sought in the organisms themselves, rather than in the kind of 
host." 

In a fourth publication of the series, Enders, Wu, and Shaffer 863 
discovered that the addition of the C Fraction of Tillett and Fran- 
cis 1409 to serum-leucocyte mixtures decreased the phagocytosis of 
both rabbit-virulent and avirulent strains of Type III by the cells 
and serum of both man and the normal rabbit. Furthermore, the 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 209 

addition of antirough pneumococcic serum to normal rabbit serum 
and cells resulted in increased phagocytosis of the strains that 
had been partially inhibited by the C Fraction ; and antirough 
pneumococcic rabbit serum protected mice against one hundred or 
more minimal lethal doses of the rabbit-virulent strain, provided 
the organisms were injected by the intravenous route. When anti- 
rough serum was absorbed with the C Fraction, the mouse protec- 
tive property was removed. In the summary and general conclu- 
sions, the authors stated: 

Since there is no evidence for the occurrence of type specific antibody 
in the normal rabbit and since, as we have shown, the Pneumococcus 
Type III whether avirulent or virulent is not removed from the blood 
stream or destroyed when the capsule is intact, the following factors 
which have been revealed in the course of our work appear to represent 
certain essential components, if not the complete mechanism, upon 
which the natural immunity of the rabbit against this organism de- 
pends, (a) The elevation of the body temperature after intravenous in- 
fection to 41 °C. or thereabouts and its maintenance for varying peri- 
ods, (b) The ability of the phagocytic cells, both fixed and mobile, to 
attack any cocci which have become vulnerable through the deteriora- 
tion of capsular integrity, (c) The adjuvant effect of an antibody, re- 
acting with the somatic C carbohydrate, which enhances the phagocyto- 
sis of such organisms as no longer possess a completely intact envelope. 

Conversely, the varying degrees of virulence for rabbits observed 
among Pneumococcus Type III strains are based upon: (a) differences 
in the ability of the organisms to multiply at the elevated temperatures 
encountered in the infected host. Strains markedly susceptible to the 
harmful influence of this factor fail to induce a generalized fatal infec- 
tion. Not all "thermo-resistant" strains are highly virulent, however, 
and these may contrast sharply with regard to (b) size of the capsule 
and the ease with which it is impaired or completely lost. The capsules 
must be maintained intact for a sufficient time until multiplication of the 
organisms can proceed to such a degree that death of the host results. 
Avirulent strains even when capable of growth at 41 °C. appear to be 
unable to satisfy this requirement. 

The differences in virulence of various strains apparently condi- 
tioned by these factors are not limited solely to the case of the rabbit, 
since for at least two strains similar differences in virulence have been 



210 BIOLOGY OF PNEUMOCOCCUS 

shown to exist when the intravenous route of infection is employed in 
mice. 

The results of the studies by Enders and his colleagues were con- 
temporaneously confirmed by the data obtained by Rich and Mc- 
Kee 1138 in an investigation on native immunity of the rabbit to 
Type III Pneumococcus. Strains of encapsulated Type III pneu- 
mococci virulent for mice, when injected intradermally into rabbits 
proliferate progressively in the tissues for some hours causing, 
with the dose used, a local lesion of considerable severity, often ac- 
companied by bacteriemia. The proliferation of the cocci is then 
checked, the organisms are destroyed, and the animal completely 
recovers. During the first twenty-four hours of the infective proc- 
ess the rabbit acquires a greatly enhanced resistance to further in- 
fection and the increased resistance is due to the fever developing 
in the animal as a result of the infection. The encapsulated strains 
to which the rabbit is resistant are not phagocyted promptly 
either in vitro or in vivo, but after some hours' sojourn in the body 
of the rabbit, the organisms gradually lose their capsules and are 
avidly phagocyted. As in the body of the rabbit, the majority of 
the strains studied exhibited marked sensitivity to temperatures of 
104° to 106° in vitro. 

The work of Enders and his co-workers and of Rich and McKee, 
therefore, offers a striking example of one type of mechanism op- 
erating in a host-parasite relationship and again emphasizes the 
dependence of virulence upon a special physiological instead of a 
strictly immunological response on the part of a given animal 
species. 

ARTIFICIAL, EXALTATION OF VIRULENCE 

There are several methods for raising the virulence of pneumo- 
cocci. One is the frequent transplantation of the culture into a fa- 
vorable medium, as advocated by Wadsworth and Kirkbride, 1471 
and by Felton and Dougherty; 411 " 2 ' 423 " 4 another is serial passage 
through suitable animals, or by inoculation of animals with an or- 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 211 

ganism of low virulence along with a killed culture of high viru- 
lence ; while another method is to grow the organism in antirough 
pneumococcic serum of the homologous type. Propagation in the 
body of susceptible animals has long been practiced for the pur- 
pose of enhancing the virulence of pneumococci. Eyre and Wash- 
bourn (1898) 374 employed the guinea pig, Truche, Cramer, and 
Cotoni (1911) 1426 the mouse, while Neufeld and Ungermann 
(1912) 1001 " 2 increased the pathogenicity of Pneumococcus, when 
administered to guinea pigs by inhalation, by passage through 
animals of the same species. 

The procedure of serial animal passage is now too well known 
to require extended comment. The intraperitoneal inoculation of 
white mice with infectious material containing pneumococci or with 
pure cultures, with subsequent recovery and cultivation of the or- 
ganism from the heart's blood of the test animal is the method com- 
monly used for raising and maintaining the virulence of pneumo- 
cocci for experimental purposes or for the preparation of vaccines 
for the immunization of horses in the production of therapeutic 
serum as well as for testing the potency of such serums. The stimu- 
lating action of the dead bodies of virulent strains of pneumococci 
when injected simultaneously with living cultures of avirulent or- 
ganisms of the corresponding serological type, resulting in the ac- 
quisition of higher virulence, has been discussed in the chapter on 
dissociation. A similar effect is produced by growth of organisms 
of low virulence in media containing immune serum specific for de- 
graded variants of the homologous type. 

DEGRADATION OF VIRULENCE 

Pneumococci as they encounter unfavorable conditions when cul- 
tivated outside the animal body rapidly lose virulence. Insufficient 
nourishment, excess of alkali or acid, unsuitable incubation or 
storage temperatures, and infrequency of transplantation even in 
a favorable medium, lower the vitality of the cell, inhibit capsule 
formation, and consequently attenuate or destroy the pathogenic 



212 BIOLOGY OF PNEUMOCOCCUS 

power of the organism. Furthermore, there are other and specific 
factors which may expedite the process. 

DISSOCIATION AND VIRULENCE 

Stryker 1348 in 1916 reported that virulent pneumococci when 
grown in homologous immune serum fail to form capsules, show a 
decrease in virulence, and become more susceptible to phagocyto- 
sis. By passing the altered forms through mice, reversion to the 
original type takes place. Normal serum has no such effect. The ex- 
periments of Stryker, therefore, constituted the first demonstra- 
tion of the ability of the specific antibodies of immune serum to 
inhibit the elaboration of capsular substance by Pneumococcus 
and consequently to render the organism vulnerable to the natural 
defenses of the animal body. Further developments in the investiga- 
tion of the action of immune serum as well as of cultural conditions 
on the virulence of pneumococci have already been described in 
such detail, that, in order to avoid needless repetition, the reader is 
referred to the chapter on dissociation. 

Summary 

To summarize the data discussed in the present chapter, it may 
be repeated that in the mouse and the rabbit man has at his dis- 
posal animals admirably adapted for the several purposes of the 
bacteriological and immunological study of Pneumococcus. The 
use of in-bred strains of mice, and presumably of rabbits, reduces 
the frequency of irregular results in experimental work, especially 
in quantitative estimations of specific antibodies in immune serum. 
The monkey, being more closely related in the biological scale to 
man, presents opportunity for studying the invasiveness of pneu- 
mococci and their pathological effects in the animal economy. The 
horse, although obviously unsuited to the study of pathogenicity, 
by virtue of its size and disposition, serves as the most convenient 
animal for the production of antipneumococcic serum. Susceptibil- 



PATHOGENICITY FOR EXPERIMENTAL ANIMALS 213 

ity or resistance depends on peculiarities due to the species, on 
genetic factors, age, weight, environmental conditions, and the 
physical state of the test animal. 

In addition to the variables in the host, the invasiveness of a 
given strain of Pneumococcus is conditioned by its serological 
type, the vital condition of the culture, its mass or density, the 
route of inoculation, and by intrinsic factors possessed by various 
strains of the organism. Virulence of a pneumococcus for a given 
animal species may be raised by serial passage through animals of 
the same species, and the enhanced pathogenicity can be main- 
tained by continued animal passage or by the application of suit- 
able in vitro methods of preservation. Contrariwise, the pathogen- 
icity of a pneumococcal strain may be decreased by subjecting the 
organisms to unfavorable cultural conditions or by propagating 
the organisms in media containing increasing amounts of homolo- 
gous immune serum. Pathogenicity or virulence, therefore, is only 
a relative term and must be interpreted in the light of the biologi- 
cal characters of the pneumococcal strain and of the functional 
variables in the animal host. As yet no in vitro test has been de- 
vised which equals the use of animals, particularly the mouse and 
the rabbit, for studying the invasive power or virulence of Pneu- 
mococcus. 



CHAPTER VII 
PATHOGENICITY OF PNEUMOCOCCUS : MAN 

The infectiousness of pneumococci of the different serological 
types for human beings; incidence and lethal powers of the cocci 
in disease; their distribution in the body and the lesions pro- 
duced; and the various phenomena of the carrier state. 

Pneumococci, if successful in passing the first defensive bar- 
riers of the human system, may become localized in a tissue or 
organ, mainly the lungs, whence they may invade the blood stream 
and under favoring conditions incite metastatic foci in other parts 
of the body ; or, after gaining entrance, they may proceed to sites 
other than pulmonary tissue and there set up primary infection. 
The nature and severity of the infection depend on the serological 
type, virulence, and mass of invading cocci, the passage by which 
they are admitted, and on the many and varied factors which con- 
stitute natural resistance or which are involved in the creation of 
specific immunity in response to the presence of the invading or- 
ganisms. 

The epidemiology and the clinical and pathological aspects of 
pneumococcal infection may be left to writers competent to discuss 
these subjects.* Therefore, assuming that the reader of this review 
is more intimately interested in the vital activities of Pneumococ- 
cus as a microorganism than in the manifold morbid manifesta- 
tions of the human body's response to its invasion, the present dis- 
cussion is directed more particularly to the ability of pneumococci 
of the different serological types to incite disease in man. 

Pneumococci frequently lead a vegetative existence in the nor- 
mal mouth, abiding there without causing any appreciable disturb- 

* A contemporary review of the literature relating to these subjects has been 
prepared by Heffron.soi 



PATHOGENICITY FOR MAN 215 

ance in the host. The organisms may be virulent or avirulent and 
they may sometimes be found as the predominant bacterial species 
but, contrary to the older opinion, these so-called normal pneumo- 
cocci, although possessed of full virulence, rarely cause pneumonia 
in the individual in whom they temporarily dwell. Since the dis- 
covery by Sternberg, 1316 " 8 Pasteur, 1065 " 6 and other early investiga- 
tors that pneumococci virulent for laboratory animals were to be 
found in the saliva of healthy persons, many reports have ap- 
peared concerning the frequency of the occurrence. Buerger 
(1905), 164 by the plate method, detected pneumococci in the 
mouths of thirty-nine out of seventy-eight normal persons. Some of 
the subjects were assumed to have acquired the organisms in hos- 
pital wards and others as a result of pneumonia. In both groups, 
the pneumococci persisted for days and even months. In a com- 
munication published at the same time, Hiss, Borden, and Knapp, 651 
from their bacteriological study of twenty-two healthy individuals, 
concluded that practically every person, at least during the win- 
ter season, living under such conditions as exist in New York City, 
acts as a host at some time or other and probably at repeated in- 
tervals for Pneumococcus. A seasonal effect on the distribution of 
pneumococci was suggested by Longcope and Fox 825 who observed 
a greater incidence of the organism in normal human beings during 
the winter months than during the milder seasons. The authors 
stated that during the winter a large percentage of healthy indi- 
viduals harbor virulent pneumococci in the buccal cavity, and that 
it is almost certain that some persons always have virulent pneu- 
mococci in their saliva. More recently, Brown and Anderson 
(1932) 152 noted a correlation between the incidence of pneumo- 
cocci in the throats of normal persons and periods of inclement 
weather. 

Park and Williams, 1058 by the mouse inoculation method, found 
pneumococci in the sputum of 55 per cent of over two hundred 
healthy subjects. McLeod, 880 in reviewing these reports, gave the 



216 BIOLOGY OF PNEUMOCOCCUS 

more conservative estimate of 20 per cent. Be that as it may, since 
the differentiation of the species into the many serological types, 
the list of publications which have appeared reveal the widespread 
prevalence, wherever investigated, of pneumococci of one type or 
another as members of the normal bacterial flora of the mouth. 
There is some doubt, however, as to how far down in the respira- 
tory tract of healthy individuals pneumococci may be found in a 
state of saprophytism. Pneumococcus under favorable conditions 
may exchange this benign vegetative existence for a life of malig- 
nity for man. According to Hiss and Zinsser — who voiced a gen- 
eral opinion — lobar pneumonia is the type of infection most often 
caused by Pneumococcus. McLeod differed and expressed the be- 
lief that "the most frequent lesion produced by the organism is a 
catarrhal condition of the upper respiratory tract involving the 
larger bronchi, associated with slight or considerable pyrexia, and 
often popularly described as 'influenza.' " The truth of the mat- 
ter is of less concern to the bacteriologist than to the clinician. On 
the other hand, the fact is incontestable that the great majority 
of cases of lobar pneumonia are caused by Pneumococcus. In 
addition, Pneumococcus may be the sole causative factor in bron- 
chopneumonia, sinusitis, otitis, mastoiditis, meningitis, peritonitis, 
nephritis, arthritis, and infections of the eye and other tissues. 
Some of the manifestations may be primary in character or 
secondary to pneumococcal disease in the lung. In company with 
organisms of other bacterial species, Pneumococcus may compli- 
cate or intensify the severity of the infection. 

Etiology of Pneumonia 

PNEUMOCOCCAL TYPES IN LOBAR PNEUMONIA 

The predominance of Pneumococcus as the causative agent in 
lobar pneumonia with the percentage distribution of other bac- 
terial species is seen in the table supplied by Heffron. 601 



PATHOGENICITY FOR MAN 



217 



Organism found 
Pneumococcus 
Streptococcus 
Friedlander's bacillus 
Influenza bacillus 
Staphylococcus 
Mixed infections 

Total 



Number 




of cases 


Per cent 


3,189 


96.1 


94 


2.8 


17 


0.5 


7 


0.2 


6 


0.2 


6 


0.2 



3,319 



100.0 



Heffron in a searching inquiry into the part played by the various 
serological types in the causation of lobar pneumonia collected all 
the available data and presented analyses of the figures obtained. 
In 14,869 cases of the disease reported from many parts of the 
world, pneumococci of Type I occurred in 32.8 per cent; Type II 
in 20.6 per cent; Type III in 10.8 per cent; while all organisms 
listed under Group IV were found in 35.8 per cent of the cases re- 
ported. The incidence of pneumococcal types in lobar pneumonia 
arranged according to geographic distribution, as far as reported, 
may be seen in the table taken from Heffron. 601 



PERCENTAGE DISTRIBUTION BY SEROLOGICAL TYPE OF 14,869 

CASES OF PNEUMOCOCCUS LOBAR PNEUMONIA IN 

CERTAIN GEOGRAPHIC AREAS 





Total 
cases 


Percentage distribution 


Geographic group 


Type I 


Type II 


Types I and 
II together 


Type 111 


Group IV 


United States, 
Puerto Rico, and 
Canada 

Europe 

Africa 


10,860 

3,138 

230 

641 

14,869 


31.5 
38.7 
19.1 

41.0 

32.8 


19.2 
28.3 
12.2 

15.4 

20.6 


50.7 
67.0 
31.3 

56.4 

53.4 


11.8 
7.0 
2.2 

6.6 

10.8 


37.5 
26.0 
66.5 


Far East and Aus- 
tralia 


37.0 


Total 


35.8 







218 



BIOLOGY OF PNEUMOCOCCUS 



There occur, of course, local and seasonal variations in the 
prevalence of pneumococcal types in lobar pneumonia but the fore- 
going table furnishes a composite picture for the world at large. 
Since the further division of pneumococci into the present thirty- 
two serological types, the figures of Sutliff and Finland, 1361 and of 
Bullowa, 183 as tabulated by Heffron, may be taken as representa- 
tive of the time and place of observation (Tables A and B). 

There is no call to include in this chapter the many further de- 
tailed analyses presented by Heffron as to the percentage incidence 
of pneumococcal types in relation to age and sex of lobar pneu- 
monia patients. It may suffice to include only a summary table 
showing the collected statistics (Table C). 



A. NUMBER AND PERCENTAGE DISTRIBUTION OF CASES OF PNEU- 
MOCOCCUS LOBAR PNEUMONIA, DUE TO TYPES I TO XX 
INCLUSIVE, IN ADULTS (SUTLIFF AND FINLAND'S 
AND BULLOWA'S SERIES) 



Type 


Sutliff and 

Finland's 

cases 


Bullowa's 
cases 


Total 
cases 


Per cent 


I 


268 


253 


521 


30.5 


II 


142 


79 


221 


12.9 


III 


121 


115 


236 


13.8 


IV 


14 


65 


79 


4.6 


V 


38 


60 


98 


5.7 


VI 


9 


29 


38 


2.2 


VII 


37 


69 


106 


6.2 


VIII 


42 


98 


140 


8.2 


IX 


14 


25 


39 


2.3 


X 


19 


8 


27 


1.6 


XI 


8 


6 


14 


0.8 


XII 


6 


14 


20 


1.2 


XIII 


4 


8 


12 


0.7 


XIV 


14 


45 


59 


3.5 


XV 


3 


1 


4 


0.2 


XVI 


1 


4 


5 


0.3 


XVII 


1 


8 


9 


0.5 


XVIII 


19 


24 


43 


2.5 


XIX 


4 


16 


20 


1.2 


XX 


6 


11 


17 


1.0 


Total 


770 


938 


1,708 


100.0 



PATHOGENICITY FOR MAN 



219 



B. NUMBER AND PERCENTAGE DISTRIBUTION OF 1,000 CASES OF 

PNEUMOCOCCUS LOBAR PNEUMONIA, DUE TO TYPES I TO 

XXXII INCLUSIVE, IN ADULTS (bULLOWa's SERIES) 



Type 


Number 


Per cent 


I to XX inclusive 


770 


77.0 


XXI 


13 


1.3 


XXII 


16 


1.6 


XXIII 


3 


0.3 


XXIV 


3 


0.3 


XXV 


2 


0.2 


XXVI 


- 


0.0 


XXVII 


2 


0.2 


XXVIII 


9 


0.9 


XXIX 


5 


0.5 


XXX 


2 


0.2 


XXXI 


3 


0.3 


XXXII 


4 


0.4 


Total 


1,000 


100.0 



C. NUMBER AND PERCENTAGE DISTRIBUTION BY AGE AND SEX OF 

TYPES I, II, III, AND GROUP IV CASES OF LOBAR 

PNEUMONIA IN ADULTS 





TOTAL 
CASES 


UNDER 50 YEARS 


OVER 50 YEARS 




Men 


Women 


Men 


Women 


PNEUMOCOCCUS 


Cases 


Per 
cent 


Cases 


Per 

cent 


Cases 


Per 

cent 


Cases 


Per 

cent 


Type I 
Type II 
Type III 
Group IV 

Total 


627 

362 
263 
615 

1,867 


462 
236 
124 
398 

1$20 


73.7 
65.2 
47.1 
64.7 

65.8 


73 
44 
42 

86 

845 


11.7 
12.2 
16.0 
14.0 

13.1 


83 

76 

70 

101 

880 


13.2 
21.0 
26.6 
16.4 

17.7 


9 

6 

27 
30 

72 


L4 

1.7 

10.3 

4.9 

3.9 



PNEUMOCOCCAL TYPES IN BRONCHOPNEUMONIA 

Quoting from Heffron : "The frequency with which the various 
types of pneumococci are found in bronchopneumonia approxi- 
mately parallels the frequency with which they are carried in the 
mouths of normal persons, which suggests that chance occurrence 
may play a large part in these infections." So short a time has 



220 BIOLOGY OF PNEUMOCOCCUS 

elapsed since the serological classification of pneumococci has been 
enlarged to comprise thirty-two types that the few type analyses 
in bronchopneumonia can only be looked upon as suggestive. In one 
of Heffron's tables listing the types of organisms found in this dis- 
ease in East Africa, Germany, Great Britain, India, and the 
United States, totalling three hundred cases, it is seen that Type I 
pneumococci were found in 6.3 per cent, Type II in 4.3 per cent, 
Type III in 14 per cent, and organisms of the fourth group (type 
not specified) in 75.3 per cent of all cases reported. There is a 
marked difference in the reported figures between the incidence of 
Types I and II combined (10.6 per cent) in bronchopneumonia 
and their incidence in lobar pneumonia (over 50 per cent). 

In observations on the presence of the various pneumococcal 
types in bronchopneumonia, Sutliff and Finland found that the 
ten types occurring in a series of 174* cases were, in order of fre- 
quency, Types III, VIII, XVIII, X, V, VII, XX, II, XI, and 
XIV. Additional data are given in the communication of Trask, 
O'Donovan, Moore, and Beebe. 1417 Studying the relation of pneu- 
mococci of the first three types to pneumonia, the latter authors 
noted that, in patients suffering from Type I or II infections, 
bronchopneumonia was rare, but with Type III this form of the 
disease constituted one-third of the affections. Sutliff and Finland 
reported that 21 per cent of the Type III and 30 per cent of the 
Type VIII infections studied were cases of bronchopneumonia, 
whereas only 3 per cent of the Type I and 5 per cent of Type II 
affections were diagnosed as bronchopneumonia. The early studies 
at the Hospital of the Rockefeller Institute pointed to extrinsic 
infection as the cause of lobar pneumonia, whereas the few data 
available seem to indicate that bronchopneumonia arises more 
commonly from intrinsic infection or auto-inoculation. 

In the case of bronchopneumonia following other affections of 
the respiratory tract, the infection is largely due to the bacterial 
species more commonly present in the air passages, among which 
the streptococci predominate. Pneumococci of the fourth, hetero- 



PATHOGENICITY FOR MAN 221 

geneous group may account for approximately 50 per cent of the 
cases. Streptococci of both hemolytic and non-hemolytic varieties 
have been found in about 31 per cent of the patients studied ; the 
Pfeiffer bacillus and pneumococci of Types I and II were respon- 
sible for 3.3 per cent each ; while Type III pneumococci appeared 
two and one-half times as often as did strains of either Type I 
or II. 

PNEUMOCOCCAL TYPES IN PNEUMONIA IN INFANTS AND 
CHILDREN 

The data presented in Heffron's analyses make it appear that, 
as far as reported, of pneumonia in children under twelve years of 
age, 9 or 10 per cent of the cases were caused by Type I pneumo- 
cocci, between 2 and 4s per cent were due to organisms of Type II 
or III, while in approximately 75 per cent of the cases pneumo- 
cocci of the remaining types appeared to be the causative organ- 
isms. Heffron placed Types I, XIV, VI, V, and VII as the order in 
which pneumococci are found in the pneumonias of childhood. 
There appears to be a conspicuous rise in the frequency of pneu- 
mococci of the first three types in pneumococcal pneumonia as age 
increases. Dividing the children studied into one group comprising 
those under three years of age and a second group in which the 
ages ranged from three to twelve years, the most marked increase 
is to be noted in the case of Type I Pneumococcus, which rose from 
4.2 per cent in the first group to 20.2 per cent in the second. For 
pneumococci of Types I, II, and III the corresponding combined 
incidence was 11.1 and 25.2 per cent respectively in the two age 
groups. The number of cases examined is too small to allow of ac- 
curate statistical deduction, but the trend is significant and points 
to the greater frequency of organisms of the first three serological 
types in the higher age groups. 

Without burdening the text with too many tables, Heffron's 
summary may be abridged to read : Among the 826 cases of pneu- 
mococcal lobar pneumonia in children of eleven years of age or 



222 BIOLOGY OF PNEUMOCOCCUS 

under, Type I organisms were found in 22.4 per cent, Type II in 
8.1 per cent, and Type III in 5.5 per cent, these three types to- 
gether accounting for 36.0 per cent of the infections. Sixty-four 
per cent of the remaining cases were due to pneumococci of the 
other serological types. Of all the various types, XIV, I, VI, V, 
and VII were the more prevalent, with IV, IX, XV, and XIX of 
frequent occurrence. 

In pneumonias of the bronchial type the picture is somewhat 
different. Whereas Type II Pneumococcus showed slightly greater 
frequency, Type I organisms fell in percentage of occurrence from 
22.4 to 9.0, and Types I, II, and III combined showed a decrease 
of from 36.0 to 26.2 per cent. The respective rates for pneumo- 
cocci other than those of the first three types were reported to be 
substantially the same for infants and children and for adults. 

To recapitulate the enumeration so far reported of the com- 
monest serological types of Pneumococcus in order of approximate 
frequency as occurring in infants, children, and adults, the follow- 
ing figures from Heffron are presented: 

Lobar pneumonia 

Infants and children . . . . XIV, I, VI, V, VII 

Adults I, II, III, VIII, VII, V 

Bronchopneumonia 

Infants and children .... VI, XIX, XVIII 

Adults Ill, VIII, XVIII, X, V, VII 

Serological Types and Fatality-Rates 

The factors making for virulence of pneumococci have been dis- 
cussed in Chapter VI. In addition to the characters of individual 
strains dependent upon their history and immediate environment, 
there are variations in invasive power peculiar to the many sero- 
logical types. The data so far accumulated on the fatality-rates 
of the different types are not extensive but enough evidence is at 
hand to warrant some general, if tentative, conclusions. Heffron 
stated that from available reports the fatality-rates, at least for 



PATHOGENICITY FOR MAN 



223 



lobar pneumonia due to pneumococci of Types I and II, are some- 
what lower in Great Britain, and possibly in Norway, Sweden, and 
Germany, than in the United States and Canada. For 1,614 civil- 
ian cases receiving no serum treatment in the two American coun- 
tries, Heffron calculated the death-rate as 25 per cent for Type I 
cases and as 41 per cent for cases due to Type II pneumococci, 
while of Type III infections from 45 to 60 per cent of the cases 
terminated fatally. 



FATALITY-RATES OF LOBAR PNEUMONIA DUE TO PNEUMOCOCCI 
OF TYPES IV TO XXXII INCLUSIVE 









Death-rate 


Type 


Cases 


Deaths 


per 100 


IV 


43 


13 


30.2 


V 


90 


27 


30.0 


VI 


18 


8 




VII 


97 


28 


28.8 


VIII 


67 


15 


22.4 


IX 


25 


8 




X 


24 


8 




XI 


12 


2 




XII 


12 


5 




XIII 


17 


4 




XIV 


20 


2 




XV 


4 


1 




XVI 


6 


- 




XVII 


3 


- 




XVIII 


30 


10 


33.3 


XIX 


11 


4 




XX 


10 


2 




XXI 


2 


1 




XXII 


- 


- 




XXIII 


1 


1 




XXIV 


- 


- 




XXV 


- 


- 




XXVI 


1 


- 




XXVII 


- 


- 




XXVIII 


- 


- 




XXIX 


- 


- 




XXX 


- 


- 




XXXI 


- 


- 




XXXII 


- 


- 




Total 


493 


139 


28£ 



Note: Rates are omitted where the numbers are too small to be significant. 



224 BIOLOGY OF PNEUMOCOCCUS 

Conning the reports published by Bullowa, Park, and Sutliff and 
Finland, Heffron arranged in tabular form the data representing 
the fatality-rate in lobar pneumonia caused by pneumococci of 
types other than the first three. 

The above data are too few to permit statistical analysis, yet 
they are already guiding manufacturers in preparing antipneumo- 
coccic serum in accordance with the more prevalent types of pneu- 
mococci responsible for lobar pneumonia in this country. 

The only report available on the lethal power of the different 
types of Pneumococcus in bronchopneumonia is that of Sutliff and 
Finland (1933). 1361 Here again, the data are meager, but there can 
be no gainsaying the fact that bronchopneumonia caused by pneu- 
mococci, with its high death-rate, is a serious affliction to man. 

Localized Epidemics of Pneumococcal Infection 

The infectiousness of Pneumococcus for individuals living in 
close association is demonstrated by the occasional outbreaks of 
acute disease of the respiratory tract. Sinigar (1903) 1292 described 
an epidemic among the staff and patients of an asylum, but inas- 
much as the bacteriological study consisted merely in the micro- 
scopic examination of preparations of sputum and from the lungs 
of fatal cases, one is in the dark as to whether the epidemic was 
caused by a pneumococcus. 

The presence of pneumococci of the various serological types 
associated with common colds has been too frequently noted to re- 
quire extended discussion. The results of a study reported by Val- 
entine (1918) 1442 may be cited as typical. From sixty-five cases of 
upper respiratory infections diagnosed as colds, pneumococci were 
recovered in forty-three instances. Of the organisms, two belonged 
to Pneumococcus Type I, two to Type II, four were of the third 
type, and the remainder fell into the heterogeneous Group IV. A 
more compact source of material for study was an outbreak of re- 
spiratory infection among the inmates of a children's home shelter- 
ing more than seven hundred children of both sexes between the 



PATHOGENICITY FOR MAN 225 

ages of two and twelve years. In investigating the 150 cases occur- 
ring among the inmates, Schroder and Cooper (1930) 1250 isolated 
an organism described by them as extremely infective and which 
they identified as Type V Pneumococcus. All the cases save one oc- 
curred among the boys, who were in frequent and close contact, the 
sole exception being a girl who had assembled with the boys in a 
classroom. 

A series of twelve cases of pneumonia and two of otitis media de- 
veloping over a period of four months among eighty-seven boys in 
an orphanage was caused by Pneumococcus of Type I. Strom, 1344 
who studied the epidemic, reported that the infection was more 
prevalent among the older boys and, furthermore, that of those 
coming in contact with the affected boys a third of the number 
were found to be carriers of the same organism. The strains iso- 
lated from the patients were more virulent for mice than were those 
recovered from the carriers. 

Infectious Processes Other Than Pneumonia 

Pneumococcus, during the pneumonic process, may migrate from 
the seat of infection in the lung to the vascular system and thereby 
be distributed throughout the body and create localized foci. Or, 
without the intermediate pulmonary lesion, the organisms may by 
direct or indirect routes gain access to vulnerable tissues. While, 
because of the abundant data, it is possible to apportion the guilt 
to representatives of the different serological types in lobar and 
bronchopneumonia, a verdict is not so easily pronounced in non- 
pulmonary infections of pneumococcal origin. Many of the reports 
appeared before the diversity of types was recognized, and since 
that era the comparative infrequency of non-pulmonary infections 
has supplied too few observations to be of any statistical value. 

Meningitis as a complication or sequel of lobar pneumonia had 
early been reported by Fraenkel, 469 Senger, 1255 Netter, 962 Lance- 
reaux and Besancon, 778 Gamaleia, 498 Meyer, 895 Ortmann, 10378 and 
others, but not until the report of Foa and Bordoni-Uffreduzzi 462 



226 BIOLOGY OF PNEUMOCOCCUS 

was any claim advanced that pneumococcal disease of the meninges 
could occur as a primary infection. Many references to clinical re- 
ports must necessarily be passed over in this review, but Harkavy's 
(1928) 589 is cited because from the one patient mentioned Type I 
Pneumococcus was isolated from the spinal fluid and in the case of 
this patient the administration of specific immune serum effected a 
cure. 

Endocarditis and pericarditis are not unusual concomitants of 
pneumonia, but the condition is nearly always secondary to the 
pneumonic process and the same organism may be responsible for 
both lesions. Thomas and O'Hara (1920) 1395 described a case of 
endocarditis in which Type I Pneumococcus was found in a vegeta- 
tion on the tricuspid valve, but the authors disclaimed that the un- 
usual site of the infection was due to any special selective affinity 
of that pneumococcal type. 

Such infections as sinusitis, parotitis, gingivitis, and otitis 
may accompany but are rarely independent of lobar pneumonia. 
Zaufal 1568 " 9 was probably the first to report primary infection of 
the ear by Pneumococcus. Arthritis is a metastatic manifestation 
of pulmonary infection and has been described by Nauwerck, 944 " 6 
Weichselbaum, 1503 " 5 and Herzog, 639 but it is improbable that ar- 
thritis or nephritis occurs except as a result of pneumococcal in- 
fection elsewhere in the body. To the list of non-pulmonary infec- 
tions caused by Pneumococcus, Neufeld and Schnitzer added 
osteomyelitis and appendicitis (Ungermann), epidydimitis and or- 
chitis, cystitis, fibrinous enteritis (Flexner, Curio), cholecystitis 
(Lenharz and others), phlegmon (Robbers), ulcus serpens and 
conjunctivitis (Axenfeld and others), erysipelas (v. Leube, and 
Reiche and Schomerus), and pyosalpinx and puerperal sepsis 
(Jacob). 

Of the several specific complications of pneumonia, infections of 
the pleura and pericardium are the most common. Endocarditis 
and meningitis are grave sequels, while, in addition to the affec- 



PATHOGENICITY FOR MAN 227 

tions already mentioned, abscesses, retinochoroiditis, and panoph- 
thalmia may develop as secondary lesions. 

Primary pneumococcal infections of the eye have been described 
by Mikaeljan (1931) 902 who, in a limited series of cases, found 
pneumococci of Types I and II in panophthalmia, ulcus cornea 
serpens, and purulent dacryocystitis, while organisms of Group 
IV were isolated from the eye in two cases of purulent conjuncti- 
vitis. 

In a communication appearing in 1931, Smeall 1293 tabulated the 
pneumococcal types encountered in a variety of affections and 
stressed the fact that the organisms most commonly found in the 
eye and accessory sinuses belonged to Group IV and that those of 
Type III and Group IV were present most frequently in acute 
otitis media and mastoiditis. It is of course not improbable that a 
similar and larger series of cases investigated in another locality 
or country might yield different results. 

Pneumococcemia 
From time to time bacteriologists have alleged that virulent 
pneumococci as well as streptococci and other pathogens are to be 
found in the circulating blood of healthy persons as well as of 
those ill with affections to which the organisms are not related. 
The validity of the claims is always highly questionable, and it is 
problematical if Pneumococcus invades the circulatory system un- 
less a specific focus of infection exists at some point in the body. 
Since the discovery by Friedlander 489 " 90 in 1884 of pneumococci in 
the blood of pneumonia patients, their presence has been recog- 
nized as a frequent accompaniment of pneumococcal disease indi- 
cating a gloomy prognosis. The literature and bedside records are 
replete with notes on positive blood cultures taken on pneumonia 
patients, but the percentage of positive cultures reported varies 
with the nature of the clinical material and the skill of the techni- 
cian. Prochaska 1111 claimed that he found pneumococci in the 



228 BIOLOGY OF PNEUMOCOCCUS 

blood of practically all of forty pneumonia patients ; Strouse and 
Clough (1910) 1347 reported positive results in 56 per cent of the 
cases occurring in a localized epidemic in which the total mortal- 
ity was only 20 per cent. Of cultures made by Lyall (1912), 840 
40.5 per cent were positive, and if those taken at the time of crisis 
or lysis, which were uniformly negative, are deducted, the figure 
rises to 53 per cent. The fatality-rate for the whole series was 26.2 
per cent, while that for the patients having pneumococci in the 
blood was 50 per cent. The great prognostic value of blood cul- 
tures in lobar pneumonia was early pointed out by Avery, Chicker- 
ing, Cole and Dochez (1917), 36 who reported positive blood cul- 
tures in 30.3 per cent of 448 cases. Of the positive cases, 55.8 per 
cent were fatal, whereas only 8.3 per cent of patients with no pneu- 
mococcemia died. The mortality among the patients harboring 
strains of Types II and III and of Group IV in the blood was 
above 50 per cent, and all cases in the series with Type III bac- 
teriemia terminated fatally. 

In a study made by Sutton and Sevier 1365 at Johns Hopkins 
Hospital in 1917 the numbers of deaths and of positive blood cul- 
tures were practically identical. The series was so small (62) that 
percentage figures are not of general significance. It was evident, 
nevertheless, that of the cases observed, positive blood cultures and 
deaths were far more frequent when the infecting Pneumococcus 
belonged either to Type I or to atypical Type II. That some or- 
ganisms of Group IV may be sufficiently invasive to enter the blood 
stream is shown by the figure of 18.2 per cent of positive blood 
cultures for cases in which a Group IV pneumococcus was the 
causative organism. 

The correlation between mortality and bacteriemia did not hold 
in the series reported by McClelland, 874 but pneumococci of Types 
I and II were responsible for the majority of the positive blood 
cultures and of the deaths. Here the fatality-rate for Group IV 
cases was only 4.2 — the same as the percentage occurrence of bac- 
teriemia. That pneumococcemia may be present in a large propor- 



PATHOGENICITY FOR MAN 229 

tion of pneumonia cases is also apparent from the work of Christie 
in England (1932), 232 who found pneumococci in approximately 
57 per cent of the blood samples cultured. Christie emphasized the 
importance of positive blood cultures as a prognostic measure, 
since all the fatalities in the series occurred in the positive group, 
whereas, in the case of the patients who had bacteriemia and recov- 
ered, convalescence was prolonged or complicated with empyema. 
Heffron discusses this feature of pneumococcal infection in 
greater detail. For the purposes of the bacteriologist the foregoing 
discussion may suffice. The presence of active bacteriemia and the 
development of infected foci in the deeper tissues bears witness of 
the ability of pneumococci at times to surmount the natural obsta- 
cles in the body and to migrate to parts distant from the original 
site of infection. 

Excretion of Pneumococci 

From diseased foci pneumococci pass into the sputum in cases 
of sinusitis, parotitis, gingivitis, and pneumonia ; into the spinal 
fluid in meningitis ; into all purulent exudates in pericarditis, em- 
pyema, peritonitis, and arthritis. The urine in approximately 38 
per cent of the cases of lobar pneumonia studied was found by 
Mathers (1916) 868 to contain pneumococci. The organisms ap- 
peared in the urine usually just before or immediately after crisis, 
and Mathers advised precautions to avoid the possible danger of 
passing on the infection, particularly in hospitals, by careless han- 
dling of the excreta of pneumonia patients. The intestinal tract is 
another exit by which pneumococci pass from the infected body. 
Rutz (1912) 1198 isolated virulent pneumococci from the feces of 
the majority of patients ill with lobar pneumonia, the organisms 
appearing as early as the second or third day of the disease. No 
pneumococci were discovered in the feces of the normal individuals 
tested. 

In studying the distribution of pneumococci throughout the 
body a word of warning may be said against placing reliance on 



230 BIOLOGY OF PNEUMOCOCCUS 

the result of cultures taken after the death of the subject. As early 
as 1905 Norris and Pappenheimer 1018 drew attention to the possi- 
ble fallacy in making deductions from bacteriological findings at 
necropsy, since organisms placed in the mouth after death were 
conveyed to and could be recovered from the lungs by culture in 
over one-half of the cases in which the post-mortem implantation 
was made. 

The Carrier State 

Pneumococci abiding in the mouth and nasopharynx of healthy 
persons — the chief portal of entrance of Pneumococcus to the 
body — owe their presence either to direct or indirect contact with 
other persons harboring the organisms in their oral or nasal cavi- 
ties, or to previous pneumococcal disease in the same individual. 
The mere presence of these pathogens in the body is insufficient to 
incite disease; it is the constitutional factors of the host which 
determine whether the invaders are to remain as innocuous mem- 
bers of the usual bacterial population or whether they are to aban- 
don their life of saprophytism and act as malignant parasites. In 
the absence of such depressing conditions as alcoholism, chilling, 
the exhaustion of fatigue, undernourishment, avitaminosis, or the 
contributory influence of previous or concomitant disease, Pneu- 
mococcus may develop none of its pathogenic potentialities. 

The upper respiratory passages of the new-born babe, accord- 
ing to Gundel and Schwarz, 575 are sterile at the time of delivery 
and may remain so for a few hours after birth. Then on the second 
or third day pneumococci may appear, the type and number de- 
pending upon the quantitative occurrence of similar organisms in 
the immediate environment, especially in the oral cavity of the 
mother. The infant as it enters and passes through childhood re- 
ceives from its fellows and elders contributions from their bacterial 
flora. In a systematic study of the incidence of pneumococci in the 
upper respiratory passages of normal persons, largely schoolchil- 
dren, Gundel (1933), 569 by repeated tests made every four weeks 



PATHOGENICITY FOR MAN 231 

for a year on over one hundred subjects, found that while the type 
of Pneumococcus usually proved to be the same in successive tests 
on a given individual, in many cases there were rapid and frequent 
changes in the types present. The relative infrequency of organ- 
isms of the first three types, so often observed in the mouth flora of 
healthy persons, is also to be noted in Gundel's report. Type I 
pneumococci occurred in 0.8 per cent, Type II in 0.4 per cent, 
Type III in 6.7 per cent, and organisms of Group IV (Gundel's X) 
were present in 60 per cent of the individuals tested successively 
throughout the year. 

In a continuation of the study, Gundel and Okura, 573 with even 
more painstaking methods, investigated the occurrence of pneu- 
mococci of more than one serological type in the same subject. 
Thirty-eight per cent of the individuals of the series tested car- 
ried organisms of two or more types, and the frequency of occur- 
rence was much greater among boys than among girls. The ap- 
pearance of new types was attributed by the authors to infection 
from without or possibly to the development of a type which had 
been suppressed by the dominance of the first type found. Gundel 
and Okura did not agree with Neufeld and Etinger-Tulczinska 984 
that infection of the nasal or buccal mucous membrane may lead to 
specific immunity against the infecting type without eliciting any 
apparent or at least definite morbid symptoms, since repeated 
serological tests failed to reveal any specific antibodies for the 
types carried for protracted periods. The explanation that the 
non-invasiveness of pneumococci existing in the normal mouth 
could be due to the fact that the organisms there undergo dissocia- 
tion into rough and, therefore, avirulent forms was opposed by 
Gundel and Schwarz, 575 who encountered no such variants in their 
investigations. This opinion was further supported by the results 
of a slightly earlier series of virulence tests on pneumococci iso- 
lated from normal and pneumonic sputums, which would seem to 
show that virulent strains exhibit few if any signs of variation in 
vivo. Gundel and Wasu 578 concluded that the vegetative pneumo- 



232 BIOLOGY OF PNEUMOCOCCUS 

cocci were not culpable in causing any ensuing pneumonia but that 
the infection always came from without. 

The disappearance of members of the first three types during 
convalescence and the subsequent appearance of organisms of a 
heterologous group was early established by Dochez and Avery 
(1915). 319 The authors stated that although pneumococci occur in 
the mouths of 60 per cent of normal individuals, the organisms are 
readily distinguishable from the highly parasitic types of pneumo- 
cocci responsible for the severe forms of lobar pneumonia — a con- 
vincing proof that infection in that disease is, in the majority of 
instances, not autogenic in nature, but is derived from some ex- 
traneous source. Dochez and Avery found further that in a high 
percentage of instances healthy persons intimately associated with 
cases of lobar pneumonia harbor the disease-producing types of 
pneumococci. In every such instance the strain isolated from the 
normal subject was found to correspond in type with that of the 
infected individual with whom the healthy person had come in con- 
tact. In conclusion, the authors stated that the existence of the 
carrier state among healthy persons and among those recently re- 
covered from pneumonia establishes a basis for understanding the 
mechanism by means of which lobar pneumonia spreads and main- 
tains its high incidence from year to year. 

The relative infrequency of pneumococci of the first three types 
in the mouths of healthy persons of varying ages who gave no his- 
tory of contact with pneumonia was also observed by Meyer 
(1920). 899 Among one hundred normal individuals, no strains of 
Type I or II (excepting an atypical II), and only three strains 
of Type III pneumococci were found. Group IV organisms were 
present in seventeen instances. In the sputum of fifty tuberculous 
patients Lyall 841 found pneumococci in twenty cases. Excluding 
one patient with a history of pneumonia in the previous year, 
pneumococci of Type I occurred once, of Type II none were found, 
of Type III three subjects harbored the organism, while pneu- 



PATHOGENICITY FOR MAN 233 

mococci of Group IV accounted for 75 per cent of the positive 
cultures. 

The studies reported by Rosenau, Felton, and Atwater, 1157 and 
by Powell, Atwater, and Felton 1106 in 1926 yielded results which, 
while differing in some respects from those of Dochez and Avery, 
confirm their thesis and throw additional light on the carrier prob- 
lem. Using a thoroughgoing technique for the isolation of pneumo- 
cocci from the mouth and throat, the Boston authors isolated 
pneumococci of Type I from four times as many subjects in con- 
tact with cases of lobar pneumonia as normal persons not thus ex- 
posed. Type III organisms were recovered in twice as many in- 
stances for every one hundred cases in the first group as in the 
second, but there was no appreciable difference in the incidence of 
Type II pneumococci. Strains belonging to Group IV occurred in 
83.5 per cent of the former and in 69.3 per cent of the latter sub- 
jects. The differences in the percentages representing the incidence 
of pneumococci of the first three types, as reported by the Boston 
and by the New York observers, may have been due to the broader 
inclusion by the Boston workers of persons in the near neighbor- 
hood of pneumonia patients. 

The rarity of pneumococci of Types I, II, and III in persons far 
removed from centers in which pneumonia is endemic has been re- 
ported by Milam and Smillie (1931). 903 In the isolated tropical 
island, St. John, of the United States Virgin Islands, the pneumo- 
cocci found in the nasopharyngeal flora of normal persons were 
avirulent, and representatives of the first three types were seldom 
present. Even city dwellers in a colder climate may enjoy com- 
parative freedom from the more common disease-producing types 
of pneumococci. In an extended investigation, embracing monthly 
examinations over a period from two months to three and one-half 
years, of 105 children and adults living in New York City, Web- 
ster and Hughes (1931 ) 1495 obtained pneumococci at one time or 
another from the nasal passages or the throat of 80 per cent of the 



234 BIOLOGY OF PNEUMOCOCCUS 

persons studied. Of the 500 strains isolated, 97 per cent proved to 
be serologically specific. The organisms formed smooth colonies 
and were for the most part avirulent for mice. Pneumococci of 
Types I and II were obtained from one and two individuals respec- 
tively on one occasion only. Type III organisms were encountered 
in nine subjects, Type VIII in nine, Types XVI and XVIII in 
three persons for varying periods in each case; and avirulent, 
atypical strains were isolated from thirteen persons on single and 
scattered occasions. The presence of the last-named organisms was 
considered by the authors as having no association with any type- 
transformation in vivo. 

The strains isolated in successive cultures from a given carrier 
were, with rare exceptions, of the same serological type and similar 
in colony morphology, virulence for mice, and other biological 
characters. The persons observed differed consistently with respect 
to the occurrence of pneumococci. Some were Pneumococcus-free, 
some were transient carriers, some periodic, and some chronic car- 
riers, and evidence was presented in the communication that these 
differences were due to variations in host-resistance. The incidence 
of pneumococci in all the individuals included in the study under- 
went seasonal fluctuations corresponding to changes in the preva- 
lence of coryza and sore throats in the same persons, an observa- 
tion reported by Longcope and Fox 825 in 1905. 

Further data on the variety of pneumococci to be found in the 
nose and throat flora of individuals selected only for the non- 
existence of pneumonic disease among them, are furnished by the 
report of Hoyle (1932) 662 who, like Webster and Hughes, carried 
out bacteriological observations on normal persons over periods of 
one, one and a half, and two years. Of the forty subjects, on one 
occasion — and the only one — Type I Pneumococcus appeared dur- 
ing an acute attack of coryza, the patient developing lobar pneu- 
monia three or four days later. Type II organisms were never de- 
tected. Type III pneumococci were isolated on four occasions, once 
late in a cold, in one case persisting for fourteen days in the mouth 



PATHOGENICITY FOR MAN 235 

flora of a carrier of Type IV, once in an individual free from other 
pathogenic bacteria, and once during an attack of mild bronchitis 
subsequent to a cold. The other pneumococci encountered belonged 
to the heterogeneous group. 

Another recent report is that of Brown and Anderson, 152 who 
found no strains of the first three types, but only those of the re- 
maining types — all of low virulence for mice — from a small series 
of normal subjects. Christie (1932), 232 in making cultures from 
the throats of nurses in a Glasgow hospital, isolated pneumococci 
from eleven of twelve nurses in the pneumonia ward but none from 
nurses in the control wards. Of the organisms found in the nurses 
exposed to pneumonia, Christie demonstrated Type I pneumococci 
four times and Type II seven times. Whether the organisms found 
corresponded to the types prevalent among the pneumonia pa- 
tients was not stated in the communication. 

The classification of carriers of Pneumococcus based on re- 
peated bacteriological examinations extending over a year or more 
as drawn up by Webster and Hughes was slightly modified by 
Bliss, McClaskey, and Long (1934). 131 After a year's study of 
young adults, the authors divided the subjects into non-carriers 
and chronic carriers, the latter group including those persons who 
intermittently exhibited pneumococci in the throat. While the so- 
called intermittent carriers might or might not yield positive cul- 
tures on repeated examination, the cultures when positive were 
consistently of the same type of Pneumococcus in any given case, 
indicating to the authors a chronic condition with constant bac- 
teriological findings only as to type. Furthermore, the authors 
considered that their demonstration added evidence in favor of the 
stability of pneumococcal types in the human body. From the 
foregoing discussion it would seem to be more logical to designate 
healthy individuals who harbor pneumococci in the nose and throat 
for short periods of time as temporary carriers and those in whom 
the organisms persist for longer periods as chronic carriers. A 
subdivision of the latter into continuous and intermittent carriers 



236 BIOLOGY OF PNEUMOCOCCUS 

would define more accurately the condition and the possible menace 
of the chronic class. 

Summary 

A summary of the main facts brought out in the foregoing dis- 
cussion may be presented thus : 

1. The great majority of cases of lobar pneumonia (circa 96 
per cent) are caused by Pneumococcus. 

2. Pneumococci of Types I and II are accountable for approxi- 
mately one-half of the cases of lobar pneumonia in all countries 
from which records are available, except Africa. 

3. The various serological types found in lobar pneumonia, as 
far as at present known, are, in order of frequency, I, III, II, VIII, 
VII, V, IV, XIV, XVIII. In children, pneumococci of the first 
three types are responsible for the disease in only 36 per cent of 
the cases, with Types XIV, I, VI, V, VII, IV, IX, XV, and XIX 
occurring most frequently in the order given. 

4. The fatality-rates of pneumococcal lobar pneumonia have 
been reported as 40 to 60 per cent for Type III cases, 41 for 
Type II, 25 for Type I, with Types XVIII, VII, and VIII next 
in order of lethal power. 

5. In bronchopneumonia, the types responsible for infection in 
order of incidence are III, VIII, XVIII, X, V, VII, XX, II, XI, 
and XIV. The data are tentative pending further information. 

6. Lobar pneumonia, although usually endemic, may, as is the 
case of other bacterial infections of the respiratory tract, appear 
in localized outbreaks, which are not the peculiar manifestation of 
any particular type of Pneumococcus. 

7. As accompaniments or sequels of lobar pneumonia, infections 
by the pneumococcus causing the primary lesion may arise in vari- 
ous locations in the body, or 

8. Primary lesions incited by Pneumococcus may develop in the 
eye, nasal, buccal, and aural cavities and adnexa, the meninges, 
and other tissues. 



PATHOGENICITY FOR MAN 237 

9. Healthy persons may harbor pneumococci of one or more 
types in the mouth and throat with no harm to themselves. 

10. The carrier state may be due a) to pneumococci implanted 
by transference from other persons or b) to organisms arising 
from pneumococcal disease within the individual. 

11. The organisms implanted by transference are largely of the 
heterogeneous types formerly classified as Group IV, except in the 
event that the donor is suffering or recovering from an attack of 
pneumonia due to the predominant types. The pneumococci that 
are autogenous in origin are of the type causing the pneumonia. 

12. The carrier state may be brief (transient), prolonged 
(chronic), or sporadic (chronically intermittent). 



CHAPTER VIII 
CHEMICAL CONSTITUENTS OF PNEUMOCOCCUS 

The preparation and properties of the protein and carbohydrate 
fractions of the pneumococcal cell, discussed in relation to their 
chemical constitution and immunological activities. 

The chemical complexity of pneumococci and the immunologi- 
cal significance of their constituents were never appreciated, 
or even suspected, until Heidelberger and Avery 606 announced the 
results of their study of the composition of these remarkable cells. 
Like many other bacteria, Pneumococcus had been neglected by 
chemists. Such information as we had was either based on assump- 
tion or had come from chance or collateral observations by bac- 
teriologists. Protein, of course, was present, protein combined 
with phosphorus being a part of all living cells. Then fats or lip- 
ids revealed their presence by the fatty acids they yielded on au- 
tolysis of the cell. Inorganic salts were elements necessary for its 
vital functions, although there was no detailed knowledge of their 
kind or quantity. The capsule, so distinctive of the species, had 
been believed to consist of mucin or some allied substance. It was, 
therefore, the systematic study of Heidelberger and Avery (1923) 
which disclosed the presence of the peculiar carbohydrates that 
give to strains of each type their special serological characters, 
that spell virulence or, by their absence, lack of virulence, and that 
determine the exact or specific immunological response the cocci 
call forth. These studies, moreover, rendered a greater service in 
revealing a new biological principle, that is, the action of sugars as 
antigens per se or in orienting the antigenic stimulus of their con- 
jugated protein, and in their exquisite action as haptens in the 
presence of homologous immune bodies. 

Carbohydrates had always been disregarded as possessing any 



CHEMICAL CONSTITUENTS 239 

immunizing properties — it was thought to be the proteins of the 
cells which performed this function — but now it seems that, in the 
case of Pneumococcus at least, the protein is merely the vehicle 
carrying the sugar that decides the immunological character of 
the saccharide-protein complex of the cell. 

WORK OF EARLIER INVESTIGATORS 

Before discussing the newer discoveries it may be permissible to 
recount briefly the observations of earlier investigators in order 
that their bearing on these questions may be borne in mind. The 
literature before 1923 is notable for its lack of reports of detailed 
or systematic investigations on the chemistry of Pneumococcus. 
Here and there one finds communications dealing with a few of the 
components of the cocci, some fragmentary, some pointing mostly 
by inference to the existence of proteins, fats, or carbohydrates. 
The first account was that of Friedlander 487 in 1883. His interest 
in the peculiar capsular material caused him to carry out a few 
simple tests to determine its nature. It was soluble in alkali, but 
insoluble in acetic or mineral acids, in alcohol, ether, or chloro- 
form, and these properties led Friedlander to believe that the cap- 
sule was composed of mucin or some related substance. 

While it was a foregone conclusion that the cell contained pro- 
tein, no direct evidence had been offered proving its existence. The 
affinity of the cellular material for aniline dyes was presumptive 
evidence, as was the increase in amino nitrogen during autolysis. 
The work of Rosenow 1169 and of Avery and Cullen 38 ' 41 proved that 
intracellular protein must be the source of the smaller nitrogenous 
molecules arising in the natural self-digestion of the cell. So it was 
with the fats ; only by their cleavage products were they recog- 
nized. 

Prior to 1917, none of the constituents of Pneumococcus had 
been isolated or subjected to chemical study. It was in that year 
that Dochez and Averv 321 discovered in cell-free filtrates of broth 



240 BIOLOGY OF PNEUMOCOCCUS 

cultures of Pneumococcus of Types I, II, and III, in human blood 
serum, in urine during the course of lobar pneumonia* and, at 
times, in the blood of experimentally infected animals, a soluble 
substance that gave a specific precipitate with antipneumococcic 
serum of the homologous type. According to the authors, the sub- 
stance was present in cultures when the organisms were growing at 
maximal rate and undergoing little or no cell death. Consequently 
its presence was not dependent upon cell disintegration, but repre- 
sented the extrusion of bacterial substance by the living organism. 
The substance was readily soluble in water, was not destroyed by 
boiling, was not digested by trypsin or urease, did not dialyze 
through parchment, and was precipitable by acetone, alcohol, 
ether, and colloidal iron. The substance was, of course, the now 
well-known soluble specific substance which has so broadened our 
conception of pneumococcal immunity. 

From a careful reading of Perlzweig's two papers (1921) 1078 " 9 
there seems to be no doubt that he was dealing with the same sub- 
stance but in impure state. From solutions of Pneumococcus in 
bile-salts, by Rowland's anhydrous sodium sulfate method, and by 
alcohol, Perlzweig precipitated an antigenic substance which he 
considered was a nucleoprotein. However, on autolyzing the whole 
organism or digesting it with proteolytic enzymes, the antigen was 
found to be unimpaired. It was not injured by boiling for five min- 
utes in neutral or slightly acid solution, but was destroyed by boil- 
ing in alkaline solution. The substance could be recovered from 
autolysates or digestion mixtures by extraction with alcohol in a 
concentration of 70 to 85 per cent, but it was not soluble in 95 to 
99 per cent alcohol. Perlzweig, 1079 and Perlzweig and Steffen, 1081 
described the antigen as being soluble in neutral, acid, and alka- 
line aqueous solutions but not in lipid solvents and, when tested 
antigenically on white mice, it apparently possessed the complete 
immunizing properties of the original pneumococcus. Because of 

* The finding recalls the observation of Preisziios (see Chapter II) who dem- 
onstrated the presence of this substance in the blood of pneumonia patients, but 
who thought it was mucin. 



CHEMICAL CONSTITUENTS 241 

its physical characters and the dependence of its heat stability on 
the hydrogen ion concentration of the substrate, the authors sug- 
gested that this antigen might be closely related to the antineuritic 
water-soluble B vitamin. 

In another communication (1925), Perlzweig and Keefer 1080 re- 
ported a method for purifying the antigen but gave no details of 
its immunological properties. From massive cultures in Huntoon's 
medium, after heat-killing and filtration, the antigenic substance 
was separated from non-antigenic material by ultrafiltration 
through collodion. Further purification was effected by precipita- 
tion at the isoelectric point (pH 4.1) with 0.1N acetic acid- 
sodium acetate buffer mixture. The precipitate was dissolved in 
water with the addition of a small amount of 0.1N sodium hy- 
droxide. The preparation contained 20 milligrams of nitrogen per 
100 cubic centimeters (based on the volume of the original solu- 
tion), which might indicate that it was made up, in part at least, 
of the nitrogen-containing specific carbohydrate of Type I Pneu- 
mococcus, the organism used as source material. Since Perlzweig 
and Keefer gave no other details, it is impossible to judge the pu- 
rity of their antigen, but Perlzweig and Steffen, 1081 in a 1923 re- 
port, considered that it existed in a loose chemical or physical un- 
ion with protein. It is to be presumed, therefore, that the authors 
had not isolated the polysaccharide in pure form, but rather the 
carbohydrate-protein compound from which some of the protein 
had been removed. 

At about the same time, Zinsser and Parker, 1583 continuing the 
study made by Zinsser 1575 on tuberculin, described an antigen ob- 
tained from Pneumococcus and other bacteria which was analo- 
gous to, if not identical with, the substance isolated by Dochez 
and Avery. 321 These "residues" or "residue antigens" were pre- 
pared in the following manner: Pneumococci were grown on agar, 
removed from the medium and dried, then shaken with salt solution 
at a pH of 9.0 to 9.4, and immediately used or neutralized and 
stored. The extracts were centrifuged, passed through a Berkefeld 



242 BIOLOGY OF PNEUMOCOCCUS 

filter, and precipitated with acetic acid in the cold. After separa- 
tion of the precipitate (phosphoprotein or nucleoprotein), acid 
was added until no more precipitate came out of solution when the 
precipitate was discarded. After filtration, the solution was acidi- 
fied and boiled for three to five minutes, then again filtered and 
neutralized. The filtrate was the residue solution. It became turbid 
on the addition of ten volumes of alcohol, and the precipitate con- 
tained the antigenic substance, although the authors suggested 
that it might have been mechanically thrown down. 

The residue gave a specific precipitate with homologous anti- 
serum but no complement fixation with equine antipneumococcic 
serum, nor did it stimulate any immune-body production in ani- 
mals injected with it. The substance, however, excited a positive 
reaction when injected intradermally into normal guinea pigs and, 
to a lesser degree, in tuberculous pigs. Its resistance to autoclav- 
ing and to boiling in alkaline solutions would seem to establish a 
close relationship to the soluble specific substance described by 
Dochez and Avery. 

Two years later, Zinsser with Tamiya 1584 extended the study of 
pneumococcal residue and pneumococcal protein. Some rabbits 
were immunized with agar-grown pneumococci washed with 2 per 
cent formalin in saline solution, and other rabbits with Berkefeld 
filtrates of pneumococci dissolved in minimal amounts of bile. The 
serum from these animals was then tested for precipitins against 
the two antigens. In the discussion of the experimental results Zins- 
ser and Tamiya expressed the opinion that the antigens consisted 
of two substances : the one obtained by treating pneumococci with 
weak alkali, the other by f ractioning the extract by acid precipita- 
tion. The one was a nucleoprotein independently antigenic, induc- 
ing antibodies which reacted only with itself and not with the resi- 
due ; the other was the residue material incapable of inducing any 
kind of antibody response but capable of reacting with antibodies 
formed by the injection of the whole bacteria. The two substances, 
therefore, although in a crude state, corresponded in their immu- 



CHEMICAL CONSTITUENTS 243 

nological reactions to the protein and carbohydrate fractions of 
Avery and Heidelberger. 

FIRST DESCRIPTION OF THE PNEUMOCOCCAL CARBOHYDRATE 

It was, however, Heidelberger and Avery who first gave definite 
and detailed descriptions of the carbohydrate fraction, or soluble 
specific substance of pneumococci. No microorganism, except per- 
haps the tubercle bacillus, has since that time been subjected to 
such thorough chemical investigation as Pneumococcus. The stud- 
ies of Heidelberger and Avery and their colleagues and those com- 
ing from other laboratories have supplied a rationale for the va- 
ried immunological and pathological behavior of pneumococci and 
established a chemical basis for understanding many of the diverse 
phenomena of immunity. 

In their first communication, Heidelberger and Avery (1923) 606 
described the isolation of the soluble specific substance of Type II 
Pneumococcus, which they concluded consisted mainly of a carbo- 
hydrate, which appeared to be a polysaccharide built up of glu- 
cose molecules and which, in a dilution as high as 1 to 3,000,000, 
gave a specific precipitin reaction with homologous immune serum. 
The method of its preparation was simple. It consisted in the con- 
centration of an eight-day autolyzed broth culture of Type II 
Pneumococcus, precipitation with alcohol, repeated resolution and 
precipitation, then a careful series of fractional precipitations 
with alcohol or acetone after acidification with acetic acid and, 
finally, repeated fractional precipitation with ammonium sulfate 
and dialysis of aqueous solutions of the active fractions. The prod- 
uct gave no biuret reaction and no precipitate with phosphotung- 
stic acid. It contained a trace of phosphorus but no sulfur and, be- 
cause 1.2 per cent of nitrogen was found to be present, the authors 
forebore to make any claim for the purity of the preparation. 

During the next few years there came from the laboratories of 
the Hospital of the Rockefeller Institute a series of papers under 
the authorship of Avery and Heidelberger and their associates, 



244 BIOLOGY OF PNEUMOCOCCUS 

Goebel, Morgan, and Neill, which contained descriptions of refine- 
ments in the methods of preparing these polysaccharides and of 
their physical, chemical, and immunological properties. Because of 
their importance the data are presented here in considerable de- 
tail ; and in order that the reader may be offered a full, connected, 
and authentic account of the results of these basic studies, the 
liberty is taken of quoting from and paraphrasing Heidelber- 
ger's 604 " 5 comprehensive discussions in 1927 of the chemical nature 
of the antigenic substances of Pneumococcus. 

The type II Pneumococcus was first studied and the fractionation 
and purification of the specific substance were followed at each step by 
means of the precipitin test. ... As the purification proceeded the 
material isolated took on more and more the properties of a polysac- 
charide, so that it became evident that a sugar derivative was at least 
the carrier of whatever might be the true specific substance itself. At- 
tempts were made to separate this hypothetical substance from the 
polysaccharide by precipitation with basic lead acetate, uranyl nitrate, 
or safranine, by adsorption with alumina and recovery from this, and 
even by specific precipitation with a large quantity of Type II anti- 
body solution (prepared by Felton's method), and recovery of the spe- 
cific substance from the immune precipitate, but these failed to effect a 
significant change in properties, even when pneumococci themselves 
were used as starting material instead of the broth culture. Attempts at 
a separation by means of certain carbohydrate-splitting enzymes also 
failed since the sugar derivative proved resistant to this type of hy- 
drolysis. Moreover, when exposed to the action of 1 : 1 hydrochloric acid 
in the cold, the substance diminished in specific activity only after re- 
ducing sugars began to appear, so that the specific substance and the 
polysaccharide, if not identical, appeared at least to be very closely 
associated. On hydrolysis the specific product yielded about 70 per cent 
of reducing sugars consisting mainly of glucose, as shown by the isola- 
tion of glucosazone and the formation of saccharic acid on oxidation. 
Other possible constituents remained unidentified. 

Marked differences were found between the soluble specific sub- 
stance of Type III Pneumococcus and the corresponding deriva- 
tive of Type II. The former proved to be the soluble salt of an in- 



CHEMICAL CONSTITUENTS 245 

soluble acid, far stronger than the Type II substance, and capable 
of being thrown out of solution by an excess of strong hydro- 
chloric acid. This property was of great use, not only in separat- 
ing the specific substance from accompanying glycogen or eryth- 
rodextrin, but also in effecting rapid purification without the use 
of ammonium sulfate and with fewer fractionations by alcohol. 
Successive lots agreed closely in their physical and chemical prop- 
erties, indicating a much more definite entity than the Type II 
product. The Type III soluble specific substance was thus isolated 
as a nitrogen-free polysaccharide. No further purification could be 
realized either by precipitation with barium hydroxide in excess, 
or by adsorption on highly active alumina. The yield of this solu- 
ble specific substance was greatly increased by adding glucose to 
the broth. 

In the case of Type I Pneumococcus, the amount of the soluble 
specific substance present in the culture fluid was relatively smaller 
than in the case of the Type II and III organisms and therefore 
more alcohol was required for its precipitation from the culture 
concentrate. Other modifications were necessitated by the insolu- 
bility of the substance at its isoelectric point (about pH 4), and 
advantage could be taken of its ability to form a precipitate with 
barium hydroxide in excess. The specific substance was reprecipi- 
tated by alcohol in the presence of hydrochloric acid and dialyzed. 
Being a weak base, it precipitated as the excess of hydrochloric 
acid was removed. The Type I soluble specific substance also ap- 
peared to be a carbohydrate, but differed in the lower percentage 
of sugar liberated on hydrolysis and, what is more distinctive, in 
containing nitrogen as an apparently essential component. 

Heidelberger, Goebel, and Avery 611 arranged a comparison of 
the distinguishing characters of the soluble specific substances of 
Types I, II, and III, and included that of the Type B Friedlander 
bacillus (to be mentioned later) in the table which is reproduced on 
the following page. 

The aldobionic acid, CnHi 9 Oi COOH, isolated from the hydro- 



246 



BIOLOGY OF PNEUMOCOCCUS 



lytic products of the specific polysaccharide of Type III Pneumo- 
coccus was shown to be a compound of glucuronic acid and glu- 
cose, combined in glucosidic linkage through the aldehyde group of 
glucuronic acid and one of the hydroxyl groups of glucose. The 
polysaccharides from the three types of pneumococci contained no 
sulfur or phosphorus and differed from the starch-glycogen group 
in giving no color with iodine and in their resistance to the usual 
carbohydrate-splitting enzymes. These polysaccharides, therefore, 
would appear to represent a new type of carbohydrate. 

SOLUBLE SPECIFIC SUBSTANCES OF THE THREE FIXED 

ANTIGENIC TYPES OF PNEUMOCOCCUS AND OF 

FRIEDLANDER BACILLUS (TYPE b) 







4-> 

c 








Highest dilu- 






13 








tion giving 






> 








precipitate 






a* 








with homolo- 






T3 






Reducing sugars 


gous immune 


Type 


[ol]t> 


< 


C H 


N 


on hydrolysis 


serum 












Calcu- 










Per Per 


Per 


lated as 










cent cent 


cent 


glucose 




I 


+300° 




43.3* 5.8 


5.0f 


28 (Galacturonic 
acid) 
(Amino sugar 
derivative) 


1:6,000,000 


II 


+74° 


1250 


45.8 6.4 


0.0 


70 Glucose 


1:5,000,000 


III 


-33° 


340 


42.7 5.3 


0.0 


75 Aldobionic 

acid, glucose 


1:6,000,000 


Friedlander 














bacillus B 


+ 100° 


685 


44.6 6.1 


0.0 


73 Glucose 


1:2,000,000$ 



* Theory for (C a H 10 O 5 )„, C, 44.4 per cent; H, 6.2 per cent. 

t Amino N, 2.5 per cent. 

J Rabbit antiserum. 

Note: From a determination of its diffusion coefficient, Babers and Goebelsi 
calculated a molecular weight of 118,000 for Pneumococcus III specific poly- 
saccharide, but Heidelberger and Kendalleis later found that this was an acci- 
dental value, related only to the salt concentration at which the diffusion was 
carried out. 



Heidelberger and Avery doubted if any of the specific sub- 
stances isolated at that time (1927) represented a definite chemi- 



CHEMICAL CONSTITUENTS 247 

cal compound. The three types of Pneumococcus chosen for study 
had, however, when grown on the same medium, yielded three dis- 
tinct carbohydrates, and successive preparations of each specific 
substance had been quite uniform whatever methods were employed 
in the process of purification and whether the preparation was de- 
rived from pneumococci themselves or from autolyzed broth cul- 
tures. Furthermore, the only one of these substances investigated 
in detail appeared to differ in structure from that of any other 
known non-nitrogenous polysaccharide. It was thought by Avery 
and Heidelberger that these and other considerations based on the 
data obtained warranted the belief that the three polysaccharides 
isolated represented the actual specific substances, stripped of at 
least a large portion of accompanying impurities, and that the 
substances did not merely represent inert material carrying an ex- 
tremely minute amount of the true specific compounds. 

The studies of Heidelberger, Goebel, and Avery also included an 
investigation of the protein portion of the pneumococcal cell. Hei- 
delberger, in the reviews already cited, 604 " 5 discussed this phase of 
the work as follows: 

When these microbes (Pneumococci) are dissolved, either with the 
aid of bile, or by repeated freezing and thawing, the resulting solution 
yields a precipitate of so-called "nucleoprotein" on acidification with 
acetic acid. While probably a mixture consisting largely of nucleopro- 
tein and mucoid, it still possesses immunological properties which dif- 
fer sharply from those of the soluble specific substance. In the first 
place, the protein is antigenic, while the soluble specific substance, 
though reacting specifically with antibodies to the highest degree, is 
non-antigenic and unable by itself to stimulate the production of anti- 
bodies when injected into animals. Moreover, the protein isolated from 
the three fixed types of Pneumococcus, or from a strain of the hetero- 
geneous group IV, appears serologically the same as that from any of 
the other types. Thus this portion of the pneumococcus protein is not 
type-specific, like the soluble specific substance, but is, rather, species- 
specific. 

Saito 1213 also prepared nucleoprotein from Type II pneumo- 
cocci which precipitated with both Types I and II serums and 



248 BIOLOGY OF PNEUMOCOCCUS 

with which active immunity could be evoked against Type II but 
not against Type I Pneumococcus. Saito admitted that the prepa- 
ration still contained traces of the soluble specific substance. 

An apparent relationship between pneumococcal antibodies and 
both the protein and non-protein fractions of Gonococcus has been 
reported by Boor and Miller (1931). 138 The protein substance — 
nucleoprotein according to the authors — in a dilution of 1 to 
1,000 gave definite precipitation with Types I and II antipneumo- 
coccic serum and in as high as a 1 to 10,000 dilution with Type 
III serum. The non-protein fraction was even more potent, react- 
ing positively, and always more strongly with Type III serum, in 
ten to one hundred times the maximal dilution of the protein frac- 
tion. 

Boor and Miller observed cross reactions between the gono- 
coccal antiserum and the C Fraction of Tillett and Francis and 
there is good reason to believe that the non-protein fraction ob- 
tained from Gonococcus and the C Fraction of Pneumococcus are 
similar if not identical. The results, because of their seeming bio- 
logical importance, deserve further study. There is a factor, how- 
ever, which should not be overlooked and that is the participation 
of agar in serological reactions, as demonstrated by Sordelli and 
Mayer 1306 in the case of other organisms grown on agar media. 

Stull (1929) 1349 furnished figures for some of the chemical con- 
stituents of Type III Pneumococcus.* A virulent strain of the or- 
ganism was grown in beef-infusion peptone glucose broth for six- 
teen to eighteen hours and centrifuged; then the sediment was 
washed in distilled water, and dried at 55°. Analyses of the mate- 
rial gave the following results expressed in percentage amounts : 
Volatile matter at 105°— 4.77; ash— 6.48 to 8.64; nitrogen— 
13.00 to 13.32 ; acid-insoluble nitrogen and acid-soluble nitrogen 

* In this connection there should be mentioned the analyses of Leineweber, 
Kautsky, and Famulener,799 which were taken to indicate the, comparative uni- 
formity of pneumococci of the four types in the amount of nitrogen, and there- 
fore of protein contained. 



CHEMICAL CONSTITUENTS 249 

— each 0.2; ammonia nitrogen — 1.0; total nitrogen of bases — 5.5; 
total nitrogen in filtrate from bases — 8.6; phosphorus — 2.92 to 
2.94 ; chlorine — 0.71 ; and sulfur — 0.32. An absolute alcohol ex- 
tract of the dried cocci contained 6.5 per cent nitrogen and one 
per cent phosphorus and was claimed to be entirely free from pro- 
tein. Water extraction removed material of high phosphorus con- 
tent, probably of the nature of nucleoprotein, as well as the soluble 
specific substance. Stull's ether, alcohol, acetone, 0.1N acetic 
acid, and 0.1N hydrochloric acid extracts all failed to give pre- 
cipitates with Type I, II, and III antipneumococcic serums. Only 
the dried cocci, the water extract, and the soluble specific sub- 
stance precipitated with Type III serum, and here the action was 
specific. 

It was Avery and Heidelberger's conception that, since Pneumo- 
coccus is an encapsulated organism, "The ectoplasmic layer of the 
cell is composed of carbohydrate material which is identical in all 
its biological characters with the type-specific substance. On the 
other hand, the endoplasm, or somatic substance, consists largely 
of protein, which is species- and not type-specific. This protein is 
possessed in common by all pneumococci, while the carbohydrate is 
chemically distinct and serologically specific for each of the three 
fixed types. The cell, therefore, may be conceived of as so consti- 
tuted that there is disposed at its periphery a highly reactive sub- 
stance upon which type specificity depends." It was also the au- 
thors' idea that, since this specifically reactive substance was 
found to be non-antigenic when separated from the other cellular 
constituents and was capable of inciting antibody formation only 
in the form in which it is present in the intact cell, it might be con- 
cluded that in the latter instance it existed not merely as free car- 
bohydrate but also in combination with some other substance 
which conferred upon it specific antigenic properties. 

A tabular representation of some of the immunological functions 
of intact pneumococci and of their principal constituents is taken 



250 



BIOLOGY OF PNEUMOCOCCUS 



from the review 605 which has been drawn upon so freely in the pres- 
ent discussion.* 



PNEUMOCOCCUS AND CELL CONSTITUENTS 



ANTIBODIES DEMONSTRABLE IN SERUM 



Material used for immunization 



Intact cells (SP)t 

Carbohydrate S+ 

Protein P§ 

Solutions, extracts containing free 
S and free P 

Suspension of intact cells and dis- 
sociated cell constituents (SP), 
free S, free P 



Effective 
antigen 



(SP) 

None 

P 



(SP), P 



+ 



+ 



Pre- 
cipitins 



+ 



+ 



Comple- 
ment 
fixation 



+ 



+ 



Speci- 
ficity 



+ 



+ 



+ 
+ 



+ 



* = Free S, as antigen, does not fix complement with immune horse serum; 

is active with immune rabbit serum. 

t (SP) = Carbohydrate and protein, combined antigen of cell. 

X S = Free carbohydrate, the soluble specific substance of cell. 

§ P = Free protein of cell. 



Heidelberger continued : 

It was evident that morphological dissolution of pneumococci is ac- 
companied by antigenic dissociation, for sera prepared from filtered so- 
lutions of disintegrated cells free of formed elements fail to exhibit any 
of the dominant type-specific properties which characterize sera ob- 
tained by immunization with whole bacteria. The injection of suspen- 
sions of pneumococci into animals induces the formation of antibodies 
against S [carbohydrate] alone or against both S and P [protein] 
separately, depending upon whether or not these suspensions contain 
only intact cells or a mixture of both intact and dissolved cell bodies. 
Since pneumococci readily undergo autolysis and dissolution, suspen- 
sions and broth cultures of these organisms almost invariably contain 
not only formed elements, but also more or less of dissociated cell con- 
stituents in solution. Therefore, use of suspensions of pneumococci con- 

* Many of the experimental data from which the table was compiled are to 
be found in the original papers by Avery and Heidelberger; Heidelberger and 
Goebel; Heidelberger, Goebel, and Avery; Heidelberger and Avery; Avery, 
Heidelberger, and Goebel; Avery and Morgan; and Avery and Neill. 



CHEMICAL CONSTITUENTS 251 

taining both intact cells and the soluble products of cell disintegration 
yields on immunization not only type-specific antibodies but antibodies 
reacting with the protein substance which is common to all pneumo- 
cocci. While the former generally predominate it is the presence of this 
protein antibody with its broader zone of activity which is responsible 
for the confusing cross-immunity reactions occasionally encountered in 
supposedly type-specific sera and which has in some instances led work- 
ers to deny the existence of three distinct antigenic types of pneumo- 
cocci. That the two sets of antibodies involved are separate and distinct 
is shown by absorption tests ; the antiprotein reaction bodies in such 
sera can be removed by absorption with the protein of a heterologous 
type without diminishing the titer of specific agglutinins for the homolo- 
gous culture or the precipitins for the specific polysaccharide of the 
corresponding type. . . . 

While it had been generally assumed that only the proteins and their 
derivatives provided the innumerable opportunities for isomerism and 
subtle changes requisite for the substances exhibiting the phenomena of 
specificity, the discovery of carbohydrates with specific properties is not 
so surprising as might appear on first thought. When one considers the 
number of asymmetric carbon atoms in the hexoses and pentoses, the 
different points of attachment of the lactone bridge, the possibility of 
a and (3-glucosidic unions at various positions in the molecule, and the 
addition of sugar acids, the analogs of amino acids, to the large number 
of sugars theoretically capable of entering into the composition of such 
polysaccharides, it becomes clear that perhaps only among the carbo- 
hydrates could another sufficiently large and protean group of sub- 
stances be found to afford the possibility of specific manifestations. 

The evidence of the lack of immunizing properties on the part of 
these specific carbohydrates shown in the table on page 250 was 
largely supplied by the experiments of Avery and Morgan. 54 Their 
attempts to immunize rabbits by subcutaneous and intravenous in- 
jections of the protein-free polysaccharides of pneumococci in con- 
siderable amounts and in repeated doses invariably failed to stimu- 
late the production of any demonstrable antibodies in the serum 
of rabbits so treated. The isolated protein, on the contrary, was 
antigenic, but the antibodies it induced reacted with the nucleo- 
protein fraction of both homologous and heterologous types. In 



252 BIOLOGY OF PNEUMOCOCCUS 

addition, these antiprotein serums did not agglutinate type-specific 
strains of Pneumococcus or react with the type-specific carbo- 
hydrate derived from them. This fact points to the capsular poly- 
saccharide of the cell as the bearer of the type-specific determinant. 
As a complement to these observations, Avery and Neill 59 added 
that intact pneumococci, possessing specific antigenic powers un- 
impaired by cultural or other procedures, gave rise to agglutinins 
for the homologous type and to precipitins for the type-specific 
carbohydrate derived from them. When, however, the cell was dis- 
rupted, the soluble cell-free constituents in the absence of formed 
elements failed to stimulate the formation of type-specific anti- 
bodies, but did induce the formation of antibodies reactive with 
pneumococcal protein regardless of the type from which the lat- 
ter was derived. The data pointed to the dissociation of the carbo- 
hydrate and protein fractions during the process of cell dissolution 
with the subsequent change in the type-specific action of the con- 
stituents, or, put in another way, to the loss of antigenic function 
of the polysaccharide portion of the cell when freed from its con- 
jugated protein. 

ISOLATION OF CARBOHYDRATE FRACTIONS 

From the results reported by Schiemann and Caspar, 1228 it 
would appear that they had isolated two carbohydrate fractions 
from Type II Pneumococcus. One of the protein-free substances 
was soluble in alcohol, gave specific precipitation with homologous 
serum, and was antigenic in that it immunized mice. The second 
fraction also gave specific precipitation but was insoluble in alco- 
hol. The preparations were made by boiling sodium taurocholate 
solutions of pneumococci with acetic acid. Both substances and the 
one obtained from a Type III strain were evidently free from pro- 
tein, since the authors could detect no nitrogen in the prepara- 
tions, and yet contrary to the results of Perlzweig and Steffen and 
of Avery and Morgan, the non-nitrogenous fractions possessed an- 
tigenic power since they produced immunity in mice. Schiemann 



CHEMICAL CONSTITUENTS 253 

and Caspar noted a difference in the microscopic appearance of 
the precipitates produced by the carbohydrates when added to im- 
mune horse and rabbit serum. The difference in the behavior of 
these two kinds of serum is manifested in other reactions with 
pneumococci and their constituents or products. The protein frac- 
tions as a rule failed to establish protection in mice, although 
one such preparation from Type II Pneumococcus did induce re- 
sistance of a low order to pneumococci of both Types I and II. 

Jungeblut (1927) 698 had quite a different idea concerning the 
chemical nature of the antigenic components of the pneumococcal 
cell. Pneumococci of the first three types were extracted with 95 
per cent alcohol and the filtrates of these extracts were tested for 
specific flocculation by a modified Dujarric de la Riviere method. 
Antipneumococcic serums of Types I, II, and III gave flocculation 
of varying intensity with homologous alcoholic antigens. The re- 
actions were species-specific and also type-specific to a high degree. 
From an examination of the experimental facts, and after a con- 
sideration of the data presented prior to this study, one is not in- 
clined to go all the way with Jungeblut in concluding that this 
serological reaction depended upon the presence of bacterial lip- 
ids in the antigens. Remembering the activity of minute amounts 
of specific carbohydrates in bringing down precipitins from im- 
mune serum, one feels that emphasis should be laid on Jungeblut's 
comment that the method of preparing these alcoholic antigens did 
not preclude the possibility that "certain impurities of protein or 
carbohydrate character may have been carried over into the ex- 
tract." 

Wadsworth and Brown 1467 reported the antigenic action of the 
ether-soluble fraction of Type I pneumococci. The preparation, 
admittedly impure, was active in binding complement in the pres- 
ence of antipneumococcic serum of all types but failed to stimu- 
late the formation of agglutinins, precipitins, protective or com- 
plement-fixing antibodies in rabbits. 

In 1928, Saito and Ulrich, 1214 following the lead of Heidelberger 



254 BIOLOGY OF PNEUMOCOCCUS 

and Avery, prepared what appeared to be a protein-free carbo- 
hydrate from Pneumococcus II and which produced specific pro- 
tection in mice. After dissolving the sediment from a twenty-four- 
hour serum-broth culture in sodium taurocholate, the authors, by a 
method much the same as that of Schiemann and Caspar, removed 
the precipitate coming down on the addition of acetic acid, and 
then separated the specific precipitable substance by alcohol. A 
concentrated watery solution of the material was then treated with 
a large amount of normal sodium hydroxide and the resulting sedi- 
ment discarded. Upon the addition of alcohol to the clear fluid a 
precipitate was obtained, which in a slightly acidified solution 
failed to give any positive protein tests, had a dextro-rotatory 
power of about +30° as compared with that of +74° for the 
Type II carbohydrate of Heidelberger, and which on boiling with 
hydrochloric acid yielded reducing substances. 

The carbohydrate preparation of Saito and Ulrich was strictly 
type-specific, giving protection to mice against a Type II culture 
but no protection against a Type III culture. It seems fair to as- 
sume that their carbohydrate preparation was not so pure as the 
corresponding preparation of Heidelberger and Avery since its 
dextro-rotatory power was less (+30° against +74°) and also 
since it contained 0.61 per cent of nitrogen. 

Schiemann 1226 employed the preparations made by Saito and 
Ulrich in an attempt to immunize rabbits, and attributed his fail- 
ure to the administration of too great dosage. In no instance was 
it possible to produce agglutinins, precipitins, or protective anti- 
bodies. He succeeded, however, in immunizing mice and in demon- 
strating protective substances in their blood. 

Schiemann, Loewenthal, and Hackenthal 1231 continued the study 
of pneumococcal carbohydrates and retested the conclusions of 
Perlzweig and Steffen. 1081 From acid and alcohol precipitates of 
dissolved Type I pneumococci extracts were made with methyl and 
ethyl alcohol. The authors reported that the carbohydrate frac- 
tion contained the larger proportion of the immunizing substance 



CHEMICAL CONSTITUENTS 255 

and that the alcoholic extracts showed varying specific immunizing 
properties, which they believed might be due to the presence of 
small amounts of the type-specific carbohydrate carried over by 
the alcohol. It was found, furthermore, that watery solutions of 
the carbohydrate still produced immunity in dilutions which no 
longer gave precipitation. 

The protein-free substance of Schiemann and his associates, 
purified by repeated alcohol precipitation in alkaline solution, 
showed +264° 10' angle of refraction with polarized light (com- 
pare +300° for Heidelberger and Avery's Type I polysaccharide), 
and with hydrochloric or nitric acids at 80° yielded no reducing 
sugars. The authors noticed the characteristic differences in the 
appearance of the precipitates obtained with specific immune 
horse and rabbit serum — the former appearing sooner and being 
coarser, while the latter were fine and formed a transparent mem- 
brane. The purified preparation actively immunized mice in doses 
of 0.001 to 0.0001 milligrams but failed to do so when injected in 
amounts greater than 0.01, or less than 0.00001 milligrams, and 
therefore acted in a narrower zone than the less pure fractions pre- 
pared by the method of Schiemann and Caspar. 1228 

In contrast to the immunizing action of the carbohydrate frac- 
tions of Pneumococcus in mice, as reported by Perlzweig and 
Steffen, Schiemann and Caspar, Saito and Ulrich, and Schiemann, 
Loewenthal and Hackenthal, the polysaccharides from Type I, II, 
and III pneumococci were devoid of any power to sensitize guinea 
pigs to these specific carbohydrates. When single initial doses of 
ten, twenty, and fifty milligrams of the preparations were injected 
the animals failed to react when given a shocking dose of one to ten 
milligrams twenty-one days later. This same shocking dose, how- 
ever, produced rapid and fatal anaphylactic shock when injected 
into guinea pigs passively sensitized with the precipitating serum 
of rabbits immunized with pneumococci of the homologous type. 
The experiment afforded evidence of a striking difference, there- 
fore, between the antigenic and haptenic action of preparations of 



256 BIOLOGY OF PNEUMOCOCCUS 

specific polysaccharides. Here again there was manifested that 
strange difference between immune rabbit and horse serum, the lat- 
ter failing to sensitize the guinea pigs to the carbohydrate. Both 
serums precipitate the homologous carbohydrate, but the horse 
serum fails to bind complement in the presence of capsular poly- 
saccharide as well as being incapable of rendering guinea pigs hy- 
persensitive. 

ISOLATION OF C FRACTION 

The existence of another and quite different carbohydrate con- 
stituent of Pneumococcus was demonstrated by Tillett and Fran- 
cis. 1409 The authors used as source material a degraded, non-type- 
specific R strain of Pneumococcus. This particular culture was 
chosen in order to minimize the presence of type-specific carbo- 
hydrate. The organisms centrifuged from a full-grown broth cul- 
ture were suspended in normal salt solution, the cells were frozen 
and thawed until dissolution was effected, and then, after the addi- 
tion of acetic acid, the solution was boiled for eight to ten minutes. 
After removal of the heavy coagulum, acidulation and boiling were 
repeated in order to remove all acid- and heat-precipitable mate- 
rial. The final water-clear supernatant fluid contained this new 
substance which Tillett and Francis called "Fraction C."* 

The C Fraction, although it is probably a nitrogenous sugar, is 
chemically distinct from both the type-specific capsular carbohy- 
drate and the somatic nucleoprotein. It exhibits no type-specificity, 
but yields a precipitate with serum of individuals ill with lobar 
pneumonia. Following crisis the reaction is no longer demonstra- 
ble. Furthermore, the precipitation of the pneumococcal Fraction 
C is not limited to the serum of individuals infected with Pneumo- 
coccus, since definite reactions can be obtained in streptococcal 
and staphylococcal infections and in acute rheumatic fever. 

In another communication appearing in the same year, Tillett 

* Tillett and Francis remarked on the similarity of this substance to Lance- 
field's "Fraction C" which she had obtained from hemolytic streptococci. 



CHEMICAL CONSTITUENTS 257 

with Goebel and Avery 1410 continued the study of the newly iso- 
lated fraction. The fraction was further purified by making the 
solution (the water-clear supernatant fluid described above) alka- 
line with sodium hydroxide and by precipitation with alcohol. The 
material was dissolved in water and again reprecipitated from 
faintly acid solution by alcohol. The procedure was repeated sev- 
eral times. The final precipitate was washed with alcohol and ether 
and freed from chlorides and was soluble in water but insoluble in 
organic solvents. The substance in solution showed a specific rota- 
tion of +25.0°, contained 5.07 per cent of nitrogen, and yielded 
30.0 per cent of reducing sugars on hydrolysis. Unlike the type- 
specific polysaccharide of Type I Pneumococcus it contained no 
amino nitrogen, but like other pneumococcal carbohydrates was 
protein-free. Tillett, Goebel, and Avery also made similar prepara- 
tions from S strains of Type II and Type III pneumococci. 

The C Fraction was non-toxic for mice in amounts up to one 
milligram, it produced no purpura, and an intravenous injection 
of three cubic centimeters of a concentrated solution elicited no 
symptoms in rabbits. Three series of seven daily injections of one 
cubic centimeter of the concentrate, with weekly rests, failed to 
produce in rabbits any precipitins for the C substance. When 
mixed with antipneumococcic horse serum of Types I, II, and III 
the somatic carbohydrate precipitated each of the three, thus ex- 
hibiting a broad species coverage but no type-specificity. Tillett, 
Goebel, and Avery concluded from chemical studies and animal ex- 
periments that Fraction C was a common constituent of all pneu- 
mococci, and that it was distinct from the specific polysaccharide 
and the so-called nucleoprotein. 

The isolation of the C Fraction was also accomplished by Hei- 
delberger and Kendall (1931 ) 617 during an investigation of the 
polysaccharides of Type (not Group) IV Pneumococcus. They also 
obtained the C substance from Type I and Type III pneumococci. 
The preparations showed a higher optical rotation, higher nitro- 
gen, and in two cases a higher reducing-sugar content on hydroly- 



258 BIOLOGY OF PNEUMOCOCCUS 

sis than reported by Tillett, Goebel, and Avery who, it should be 
remembered, prepared their material from rough pneumococci. An 
analysis of the C Fraction showed that it contained 4 per cent of 
phosphorus, which appeared to be firmly bound in organic combi- 
nation, thus making the C Fraction the first phosphorus-contain- 
ing specific polysaccharide to be encountered. 

RELATION OF THE PNEUMOCOCCAL CARBOHYDRATE TO CARBO- 
HYDRATES ISOLATED FROM OTHER ORGANISMS 

In addition to the C Fraction, Heidelberger and Kendall iso- 
lated from autolyzed cultures of Type IV Pneumococcus a type- 
specific carbohydrate differing markedly from those of Type I, 
II, and III pneumococci, and representing a kind of substance 
hitherto not observed among specific polysaccharides. The authors 
also obtained a chemically similar carbohydrate without specific 
function. These substances were far more difficult to separate from 
accompanying protein degradation products than the specific 
polysaccharides of Type I, II, and III pneumococci. Because of 
the difficulty of separation, the preparations probably contained 
more or less of the accompanying specific carbohydrates, but were 
in sufficiently pure state to reveal separate identities. 

The serologically inactive fraction had the lowest optical rota- 
tion and the highest carbon content of the three, was the weakest 
acid, the least soluble in alcohol or acetic acid, and differed from 
the specific Type IV carbohydrate and the C Fraction in yielding 
on hydrolysis crystals with the optical rotation of glucosamine. 
The Type IV specific substance, on the other hand, differed from 
the others in being the poorest in nitrogen and the richest in re- 
ducing sugars on hydrolysis, and in occupying an intermediate 
position as regards optical rotation, carbon content, and acidity. 
The species-specific C polysaccharide was highest in optical rota- 
tion and in total and amino nitrogen, and the poorest in reducing 
sugars yielded on hydrolysis, in carbon content, and in its propor- 



CHEMICAL CONSTITUENTS 259 

tion of acetylated nitrogen. It differed from the fully acetylated 
Type IV substance in being broken down by nitrous acid, and dif- 
fered from all other known specific polysaccharides of Pneumococ- 
cus in containing phosphorus. 

From this comparative study of the various specific carbohy- 
drates of Pneumococcus it would seem that these substances fall 
into two sharply defined groups: on the one hand, the Type II 
and Type III type-specific polysaccharides, which are nitrogen- 
free, and on the other, the Type I, Type IV, and C Fractions 
which contain nitrogen. The two last-named examples in this 
group, with their content of acetylated nitrogen, are more closely 
related to chitin than is the Type I substance. In the nitrogenous 
group, the Type I substance differs sharply from the others in its 
high optical rotation, its pronounced amphoteric character, its in- 
solubility at the isoelectric point, its freedom from acetyl groups, 
and in its high proportion of nitrogen susceptible to attack by 
nitrous acid. The C Fraction differs from the other members of 
both groups in its phosphorus content, but resembles the Type I 
substance in that its specificity is destroyed by nitrous acid ; it is 
somewhat similar to the Type IV substance in that a part of the 
nitrogen is acetylated. The Type IV soluble specific substance, on 
the other hand, differs from the Type I polysaccharide and the C 
Fraction in containing only acetylated nitrogen and in yielding as 
high a percentage of reducing sugars on hydrolysis as do the 
nitrogen-free Type II and Type III capsular polysaccharides. 

The study of the Type IV specific polysaccharide has brought 
to light a carbohydrate of a new type among those with specific 
properties. It has, moreover, again been shown that in the closely 
related pneumococcal types thus far studied, each polysaccharide 
responsible for type-specificity is different from the others in 
structure, composition, and properties. The study has also demon- 
strated the presence in Pneumococcus of a serologically inactive 
polysaccharide closely related to chitin. This observation points 



260 BIOLOGY OF PNEUMOCOCCUS 

toward the cause of the still existing uncertainty as to whether 
chitin is a constituent of the bacterial cell wall.* 

In addition to these specific pneumococcal polysaccharides, Hei- 
delberger, Goebel, and Avery 611 " 2 isolated from the B strain of the 
Friedlander bacillus a carbohydrate closely resembling that of 
Type II Pneumococcus, the similarity extending even to precipi- 
tation with Type II antipneumococcic serum. But, although there 
was great constancy in the properties of these two substances, the 
absorption of agglutinins and precipitins was not reciprocal with 
the two organisms. It was believed that the cross-relationship was 
due to the occurrence in the specific polysaccharides of both micro- 
organisms of the same or similar chemical grouping. Other strains 
of the bacillus showed no such reciprocal relations, nor was there 
any such relation between the E strains of the Friedlander group 
and Type I and III pneumococci.f 

Heidelberger, in the review already quoted, argued that if the 
fact that bacteria possess mutual absorption capacity be accepted 
as the criterion of their antigenic identity, then the failure of the 
Friedlander bacilli to exhibit this property might be taken as fur- 
ther evidence of the lack of identity of the substances involved. The 
discussion continued: 

However, granted a chemical difference between the two specific sub- 
stances, it becomes necessary to account for their marked immunological 
similarity. In the absence of further evidence as to the structural rela- 
tions of the two polysaccharides it seems reasonable to assume that 
both contain in a portion of the complex molecule the same or a closely 
similar configuration of atoms. The essential similarity in molecular 
grouping would then determine the immunological similarity of the two 
substances. In the case of Pneumococcus it has been shown that the 
polysaccharides by themselves are not antigenic, and it is believed that 

* The foregoing paragraphs are based on the discussion in the paper by 
Heidelberger and Kendall.eir 

t In this connection it may be mentioned that in the next year Julianelle6S4 
classified, by their serological reactions, strains of Encapsulate pneumoniae 
(Friedlander's bacillus) into three types, A, B, C, and one group X. 

It may interest the reader to know that in 1935 Yen and KurotchkinissD by 
means of electrolysis were able to isolate the specific carbohydrate of the Fried- 
lander bacillus, apparently free from protein and in a highly antigenic state. 



CHEMICAL CONSTITUENTS 261 

they become antigenic only when attached to some other substance, pos- 
sibly the protein of the cell. The type specific character of the anti- 
genic response, however, is dependent almost entirely upon the nature 
of the polysaccharide and not upon the substance to which it is at- 
tached. Therefore, since the specific carbohydrate of the Friedlander 
bacillus (type B) and that of Type II Pneumococcus exhibit similar 
chemical properties the antigenic response to each may also be similar 
even though the proteins or other substances with which they are com- 
bined are quite dissimilar. 

In 1929, Heidelberger, Avery, and Goebel 608 described the isola- 
tion of a soluble specific substance from gum arabic (gum acacia). 
From this gum, which would appear to have no biological relation- 
ship to Pneumococcus, by partial hydrolysis a carbohydrate was 
obtained that was comparable to the specific pneumococcal poly- 
saccharide in its precipitating activity with both Type II and 
Type III serum. The fraction on hydrolysis yielded galactose and 
two more complex sugar acids, one of which was later shown by 
Heidelberger and Kendall 617 to be aldobionic acid and glucurono- 
galactose analogous to the compounds isolated from the specific 
polysaccharide of Type III Pneumococcus. 

Another manifestation of heterogenetic specificity was that of 
the encapsulated strains of Escherichia coli, studied by Barnes and 
Wight. 87 The organism, isolated from a mouse during a type- 
determination test on pneumonic sputum, was agglutinated by 
Type I antipneumococcic horse serum, but not by a similar serum 
produced in a rabbit. The serum from rabbits immunized with this 
colon bacillus agglutinated the homologous organism and precipi- 
tated the soluble specific substance, but failed to cause agglutina- 
tion of Type I pneumococci or to precipitate Type I pneumo- 
coccal polysaccharide. In this case the connection was somewhat 
analogous to that between Type II Pneumococcus and the later 
Type B Friedlander bacillus of Julianelle. 

Zozaya 1588 published an account of the cross and quite heterolo- 
gous serological reaction with antipneumococcic serum of dextran, 
the synthetic polysaccharide produced from saccharose by Leuco- 



262 BIOLOGY OF PNEUMOCOCCUS 

nostoc mesenteroides. With serum produced in rabbits in response 
to injections of typical and rough strains of Types I and II Pneu- 
mococcus, dextran in a dilution of 1 to 5,000 gave precipitates 
with both anti-S and anti-R serums in low dilutions (1 to 2 to 1 to 
16), showing a somewhat stronger action with the latter. When the 
serums were first absorbed with the type-specific polysaccharide or 
the C Fraction of Tillett and Francis, and then mixed with dex- 
tran, the serum precipitated to the same degree as before absorp- 
tion. The outcome of the experiment would argue for the existence 
of a distinct antibody produced by the active group of the specific 
polysaccharide, which is similar to the active group of the dextran 
polysaccharide. 

Since the discovery of the soluble specific substance in Pneumo- 
coccus by Dochez and Avery in 1917, similar complex carbohy- 
drates have been demonstrated as components of several bacterial 
species. Toenniessen 1413 was the first to isolate the nitrogen-free 
polysaccharide from Friedlander's bacillus, which was later studied 
by Kramar, 753 but failed to connect it with the immunological be- 
havior of the organism. A related substance was recovered from a 
strain of the same organism by Mueller, Smith, and Litarczek, 937 
from yeast by Mueller and Tomcsik, 938 and from an encapsulated 
colon bacillus and Bacillus aerogenes strains by Tomcsik. 1414 A 
specifically precipitating, non-nitrogenous carbohydrate has been 
isolated from tubercle bacilli by Laidlaw and Dudley, 771 and a 
similar polysaccharide as well as a specifically reacting substance 
was derived by Mueller 936 from the same bacilli by fractionation 
with alcohol. 

In a preliminary report of a recent study, Kulp and Borden 764 
described the successful immunization of mice against Type I 
Pneumococcus by vaccination with a living culture of Alkaligenes 
viscosus. The authors suggested that there might be an antigenic 
relationship between the capsular material of these two organisms, 
but they had obtained no confirmation of this possibility. There is 
no doubt that other bacterial species contain analogous polysac- 



CHEMICAL CONSTITUENTS 263 

charides which may be found to possess similar chemical and im- 
munological relationships to those of Pneumococcus but their ex- 
istence in no way detracts from the soundness of our present 
serological classification of pneumococci. Tempting as it is, fur- 
ther discussion of the immunological significance of the carbohy- 
drates from sources other than Pneumococcus would not be per- 
tinent to the present subject. 

FUNCTION OF SUGARS IN DETERMINING ANTIGENIC SPECIFICITY 
AND CONJUGATED PROTEINS 

The newly discovered and highly important function of sugars 
in determining the antigenic specificity of conjugated proteins was 
studied by Goebel, Avery, and Tillett. The two first-named au- 
thors, 521 aiming to gain more exact information concerning this 
specific action of carbohydrates, set about building two isomeric 
carbohydrate-protein compounds. They first synthesized p-amino- 
phenol (3-glucoside and p-aminophenol (3-galactoside and then cou- 
pled these hexosides with the globulin from horse serum. The two 
protein-sugar complexes thus obtained differed only in the carbo- 
hydrate radical of each and in the spatial configuration of the H 
and OH groups by a single carbon atom. In a second paper Avery 
and Goebel, 44 after coupling the diazophenol glucosides to crystal- 
line egg albumin in addition to serum globulin, studied the im- 
munological behavior of the preparations. When two chemically 
different carbohydrate derivatives were bound to the same protein, 
the newly formed antigens exhibited distinct immunological speci- 
ficity. When the same carbohydrate radical was conjugated with 
two chemically different and serologically distinct proteins, both 
of the sugar-proteins thus formed acquired a common serological 
specificity. Therefore, simple differences in the molecular configu- 
ration of the two isomers — glucose and galactose — sufficed to 
orient specificity when the corresponding glucosides were coupled 
to the same protein. The unconjugated glucosides, although them- 
selves not precipitable in immune serum, specifically inhibited the 



264 BIOLOGY OF PNEUMOCOCCUS 

reaction between the sugar-protein and its homologous antibody. 
Furthermore, the sugar derivatives unattached to protein exhib- 
ited some of the properties of carbohydrate haptens ; they were 
non-antigenic in that they incited no antibody production, but 
were specifically reactive, as shown by inhibition tests, with anti- 
bodies induced by proteins containing the homologous diazotized 
glucoside. 

Tillett with Avery and Goebel 1407 tested these artificial, conju- 
gated carbohydrate-proteins for other evidence of antigenic action 
by means of experiments in active and passive anaphylaxis. The 
experiments demonstrated the capacity of the synthesized sugar- 
proteins to produce hypersensitiveness. The fact that guinea pigs, 
passively sensitized with antigluco-globulin serum, or actively sen- 
sitized with gluco-globulin, could be subsequently shocked with 
gluco-albumin, and since the same specific relations held in the pro- 
duction of anaphylaxis with galacto-proteins, it was evident that 
the antigen-antibody specificity in these instances was directly de- 
pendent upon the carbohydrate fraction of the antigenic com- 
pounds. 

In addition to the new specificity which the carbohydrate radi- 
cal conferred upon the conjugated proteins, the uncombined gluco- 
sides by themselves also exerted a definite influence on the reac- 
tivity of sensitized animals. The injection of the glucosides into 
guinea pigs sensitized with the homologous gluco-protein immedi- 
ately before the introduction of the toxigenic sugar-protein, com- 
pletely but only temporarily protected the animals from shock. 
Tillett, Avery, and Goebel found, also, that anaphylactic shock 
could be induced by uncombined globulin in guinea pigs passively 
sensitized with either antigluco-globulin serum or antigalacto- 
globulin serum, thus demonstrating that the reactions elicited by 
globulin alone were dependent upon the common protein present in 
the antigens, and were manifestations only of species-specificity. 

Continuing the investigations, Goebel and Avery 522 prepared the 
p-amino and p-nitromonobenzyl ethers of the polysaccharide of 



CHEMICAL CONSTITUENTS 265 

Type III Pneumococcus, which they succeeded in coupling with se- 
rum globulin. Avery and Goebel 45 then reported the important and 
fundamental observation that this artificial, specific carbohydrate- 
protein complex was able to immunize rabbits against infection 
with virulent Type III pneumococci, and that the serum of rabbits 
thus immunized contained type-specific antibodies which precipi- 
tated the Type III capsular polysaccharide, agglutinated Type 
III pneumococci, and specifically protected mice against Type III 
infection. From the results it would seem that the antipneumo- 
coccic response produced by an antigen known to contain but a 
single component of the pneumococcal cell indicates the unity of 
the antibodies participating in the type-specific reactions of pre- 
cipitation, agglutination, and protection, and relates the speci- 
ficity of these antibodies to that of the capsular polysaccharide as 
the reactive part of the antigenic molecule. 

ISOLATION OF "a SUBSTANCE" 

What appeared to be a new element among the specific polysac- 
charides was introduced by Enders 358 in 1930. In autolytic prod- 
ucts of Type I Pneumococcus a substance was found, thought to 
be other than the soluble specific substance, which reacted spe- 
cifically with immune serum as determined by the precipitin reac- 
tion or, in vivo, by the anaphylactic behavior of appropriately 
sensitized guinea pigs. Enders prepared autolysates by incubating 
for seventy-two hours the phenolized, centrifuged sediment from 
twenty-four-hour dextrose broth cultures of Type I pneumococci. 
He obtained the nucleoprotein by precipitating with dilute acetic 
acid the solution derived by dissolving pneumococci with bile ac- 
cording to the method of Avery and Heidelberger, while the spe- 
cific carbohydrate was prepared from pneumococci grown in dex- 
trose hormone broth according to the methods described by 
Heidelberger and his associates. 

Enders prepared a "normal" antipneumococcic serum by inject- 
ing rabbits intravenously with cultures of Type I Pneumococcus 



266 BIOLOGY OF PNEUMOCOCCUS 

grown in broth containing rabbits' blood and killed by the addi- 
tion of formalin. He obtained an immune rabbit serum by treat- 
ing the animals intravenously with repeated injections of the au- 
tolysate and of the killed hormone broth-rabbit blood cultures and 
this serum was designated "Anti-A" serum. In addition, to other 
rabbits Enders gave injections of formalinized saline suspensions 
of agar-grown pneumococci. When the Anti-A serum was tested 
against SSS* no precipitate was formed, but with an autolysate 
from smooth Type I Pneumococcus the serum was definitely pre- 
cipitating, and the reaction was type-specific. Furthermore, both 
the Type I autolysate and the supernatant fluid from the ace- 
tic acid precipitation of the autolysate gave precipitates with 
Anti-A serum but none with antipneumococcic serum prepared 
from rough strains. 

By the method devised by Ward, Enders then added to his 
"normal" serum specific carbohydrate until precipitation was com- 
plete. The centrifuged supernatant fluid no longer gave any visible 
precipitation with SSS but, when the homologous autolysate was 
added, an abundant, flocculent precipitate was observed. Inasmuch 
as the SSS had apparently exhausted its specific antibody from 
the immune serum, the appearance of a precipitate on the addition 
of autolysate indicated to Enders the presence in the autolysate of 
a precipitinogen other than the specific polysaccharide and, ac- 
cordingly, to this hypothetical substance he gave the name, "A 
Substance." 

There are two other characters of the A substance of Enders 
which should be mentioned. One was instability when exposed to 
heat. Boiled for one-half hour at pH 9 its effectiveness as a pre- 
cipitating antigen was reduced at least one thousandfold, while the 
soluble specific substance under similar treatment remained un- 
changed. When, however, the reaction was adjusted to pH 4 with 
10 per cent acetic acid, a solution containing the A substance 

* For convenience, the soluble specific substance or type-specific polysaccha- 
rides of Pneumococcus will hereafter be frequently referred to as SSS. 



CHEMICAL CONSTITUENTS 267 

could be boiled for the same period of time without losing its ac- 
tivity. In both acid and alkaline solution, autoclaving for one hour 
at fifteen pounds pressure practically destroyed its effectiveness. 
The second character was its resistance to pepsin-hydrochloric acid 
mixture. The preparation probably resisted also the digestive ac- 
tion of trypsin, but the presence of alkali in such a substrate, by 
itself, tended to destroy the substance and therefore interfered 
with any accurate determination of its digestibility. 

Enders 359 extended the investigation to embrace the carbohy- 
drates of Type II and III pneumococci and, in addition to precipi- 
tative and anaphylactic methods, employed the agglutination and 
agglutinin-absorption reactions for a further comparison of his 
new antigen with the specific polysaccharide. He reported that 
type-specific agglutination of Type I, II, and III pneumococci oc- 
curred in homologous antiserum from both rabbit and horse to ap- 
proximately the same titer after the antibody reacting with the 
purified specific carbohydrate had been removed. This fact sug- 
gested that in pneumococci there exists a type-specific agglutino- 
gen which was to be distinguished from the specific carbohydrate. 
A side comment of Enders was that the presence of a specific 
agglutinogen in Pneumococcus Type II unrelated to the specific 
carbohydrate would account for the failure of this organism to re- 
move agglutinins from B. Friedldnderi Type B antiserum, and 
would add additional evidence pointing to dissimilarity of the A 
substance and the soluble specific substance as well as to the lack 
of serological identity of Pneumococcus II and Friedlander bacilli 
of the B type. 

An effect analogous to that described by Enders was reported 
by Ward 1483 in 1932. In a five-day broth culture of Type III Pneu- 
mococcus he found a type-specific substance with a powerful anti- 
bactericidal action. In comparison with the soluble specific carbo- 
hydrate, its precipitating action was far greater, requiring a much 
larger amount of antipneumococcic serum for neutralization. A 
similar substance, but in higher concentration, was also found in 



268 BIOLOGY OF PNEUMOCOCCUS 

the filtrate of a lung obtained at necropsy from a Type III pneu- 
monia patient. Tested by his whole-blood method, Ward found 
that a specimen of Type III convalescent blood, though compara- 
tively weak in anticarbohydrate antibody, was better able to neu- 
tralize the broth filtrate and lung filtrate than a corresponding 
mixture of normal blood and antiserum. Two other specimens of 
Type III convalescent blood neutralized the Type III broth fil- 
trate. Ward attempted no isolation of this precipitinogen but 
concluded: "The possibility that the reacting substance in the au- 
tolysate is more complex and less stable than the carbohydrate — 
perhaps a substance intermediate between the antigenic carbohy- 
drate compound in the intact pneumococcus and the carbohydrate 
itself — was forced on the author as the most likely explanation." 

Evidence which might be taken as indicating the existence of the 
A substance of Enders, or of some similar principle in Pneumo- 
coccus, is to be found in the work of Sabin (1931), 1204 who, like 
Enders, but independently,* attempted to exhaust potent anti- 
pneumococcic horse serum of protective antibodies by preliminary 
saturation with homologous SSS and found that in spite of this 
absorptive treatment the serum still contained an appreciable 
quantity of the specific protective substance. There appeared to be 
a lack of proportionality between the quantity of SSS used for 
precipitation and the amount of protective antibody left in the 
supernatant fluid. In the reaction some of the protective antibody 
was evidently lost, since the sum of the precipitated and free anti- 
bod} 7 represented only from 20 to 50 per cent of the total. This 
large loss might be accounted for if an excess of specific carbohy- 
drate were added since it would exert the inhibiting action ob- 
served by Felton. The same results as those obtained in the in vitro 
experiments were noted in in vivo tests in the rabbit. f 

Sabin concluded that in antipneumococcic serum there was a 

* Enders' paper was received for publication in May, 1930, and published in 
August of the same year, while Sabin's communication was accepted in Sep- 
tember, 1930, and appeared in January, 1931. 

t Incidentally, an observation of interest was that in the combination of SSS 
with its homologous precipitin there occurred a phenomenon similar to the 



CHEMICAL CONSTITUENTS 269 

type-specific antibody which was not neutralized by SSS and which 
was apparently distinct from the anticarbohydrate precipitin. 
Reasoning that this non-precipitable antibody could not have been 
evolved in response to any antigenic stimulus from the soluble spe- 
cific substance, Sabin sought an answer to the question, "Is there 
another type-specific antigen in the Pneumococcus in addition to 
the SSS?" After adding to Type I antipneumococcic serum the 
required amount of SSS for complete precipitation, he centrifuged 
the mixture after water-bath and ice-box incubation, and treated 
portions of the supernatant liquid with a 50 and 100 per cent ex- 
cess of SSS, with a saline suspension of heat-killed Type I pneu- 
mococci, and with a similar suspension of heat-killed Type II 
pneumococci. The experiment showed that the residual protective 
antibody in the supernatant fluid was not neutralized by SSS or by 
absorption with Type II pneumococci, but was definitely absorbed 
with the homologous Type I pneumococci. Sabin assumed, there- 
fore, that the neutralization was specific, and unless SSS in the 
organism was capable of neutralizing the antibody which SSS in 
solution could not, some other substance in Pneumococcus must be 
the responsible agent. This proviso, however, is all important in 
this connection. There is no proof that the SSS as prepared by 
Sobotka for Sabin, even though by his tests it was practically 
identical with the soluble specific substance of Heidelberger and 
Avery, was identical with the specific polysaccharide as it exists 
in the pneumococcal cell. 

In 1931, Wadsworth and Brown 1466 reported the isolation of a 
carbohydrate which appeared to be analogous to the soluble spe- 
cific substance and which yet more closely resembled the A sub- 
stance of Enders. The original source material was a virulent 
Type I strain, and the method consisted in removing the organ- 
isms from fifteen-and-one-half hour broth cultures by means of the 

Danysz effect in the combination of toxin and antitoxin. More SSS was required 
for complete precipitation when the total quantity was added at once, than 
when it was added in fractions of the total on successive days. This effect had 
also been observed by Heidelberger and Kendall in their studies on the precipi- 
tin reaction. 



270 BIOLOGY OF PNEUMOCOCCUS 

supercentrifuge and washing the sediment in distilled water. The 
supernatant fluid and washings were evaporated over the free 
flame to small volume and then used for the isolation of the specific 
carbohydrate by the method of Heidelberger and Avery. The 
preparation contained about 5 to 6 per cent of nitrogen. Its solu- 
tion gave negative biuret and xanthoproteic tests, a positive Mo- 
lisch test, did not reduce Fehling's solution until after hydrolysis 
with hydrochloric acid, and was precipitated by phosphotungstic 
acid. The material was more readily soluble in water than the 
polysaccharide of Heidelberger and Avery. 

When tested against antipneumococcic serum, the dissimilarity 
between the substance prepared by Wadsworth and Brown and the 
soluble specific substance of Heidelberger and Avery and its simi- 
larity to the A substance of Enders became evident. It precipitated 
specifically in Type I serum in a dilution of 1 to 6,000,000, but in 
a dilution of 1 to 600,000 it gave an immediate ring reaction with 
Type I serum which had been completely absorbed with a highly 
purified preparation of the soluble specific substance. Wadsworth 
and Brown did not test the supernatant fluid from the serum pre- 
cipitated with SSS for protective antibody. 

Again differing in properties from the Heidelberger and Avery 
polysaccharide, the substance induced immunity in mice, as dem- 
onstrated by the protection test. The immunizing effect was type- 
specific. The substance fixed complement in the presence of Type I 
antipneumococcic rabbit serum and while it caused no reaction 
when injected intravenously in a dose of one milligram into a nor- 
mal guinea pig it evoked fatal anaphylactic shock in guinea pigs 
passively sensitized not only with Type I antipneumococcic rabbit 
serum but with the same serum after removal of precipitin by ab- 
sorption with the soluble specific substance. Wadsworth and 
Brown concluded that the substance they had isolated from the 
pneumococcal cell corresponded to that of Schiemann and his co- 
workers, and to the A substance of Enders, but was distinct from 
the soluble specific substance of Heidelberger and Avery. 



CHEMICAL CONSTITUENTS 271 

In another communication, Wadsworth and Brown 1468 reported 
on chemical and immunological studies of carbohydrate fractions 
separated from pneumococci of the first three types and from an 
atypical strain as well. The cultures used were a virulent Type I 
Neufeld strain, a strain each of virulent Types II and III, and an 
attenuated Type I Pneumococcus. The carbohydrates resembled in 
character and action the substance described by Schiemann and 
his colleagues. The preparations differed in essential respects from 
the soluble specific substance of Heidelberger and Avery, so Wads- 
worth and Brown, for the purpose of distinguishing their sub- 
stance from SSS, gave to it the somewhat ambiguous name, "Cellu- 
lar Carbohydrate." 

In a 1 to 300 dilution none of the preparations gave biuret, 
Millon's, or xanthoproteic tests ; the Molisch test was positive and, 
after hydrolysis, Fehling's solution was reduced. The results of 
the micro-analysis of the "cellular carbohydrates" are shown in the 
original table taken from the paper by Wadsworth and Brown. 
The figures represent minimums and maximums. One specimen from 
each of the type preparations gave a trace of sulfur ; the remain- 
der gave none. 

The apparent presence of phosphorus in organic combination in 
all these preparations revealed a distinct and, to the authors, pos- 
sibly an important chemical difference between the antigenic and 
non-antigenic carbohydrate. The presence of amino nitrogen and 
of only small amounts of phosphorus further distinguished the cel- 
lular carbohydrates from the C Fraction of Tillett and Fran- 
cis. Unlike the SSS of Types II and III, the cellular carbohy- 
drates of these types contained nitrogen and phosphorus, although 
nitrogen had never been found in analogous preparations by Hei- 
delberger and phosphorus had been shown by Heidelberger and 
Kendall to be a constituent only of the C Fraction. These unusual 
results and the wide variations in the analytical figures lead to the 
surmise that these preparations of cellular carbohydrates were 
impure. 



272 



BIOLOGY OF PNEUMOCOCCUS 



MICRO-ANALYSIS OF THE CELLULAR CARBOHYDRATES OF THE 
PNEUMOCOCCUS 



Type of cellu- 
lar carbo- 
hydrate of 

Pneumococcus 


Prepara- 
tion 
number 


Sulfur 


Phos- 
phorus* 


Total 
nitrogenf 


Amino 
nitrogen^ 


Ash 


Water 








Per cent 


Per cent 


Per cent 


Per cent 


Per cent 


I 


6 




0.43 


4.28 




0.90 


13.52 


I 


9 




0.39 


4.37 


2.14 


0.43 


9.51 


I 


12 


Trace 


0.35 


4.01 


2.03 


0.85 


13.73 


I 


13 




0.77 


4.59 


1.91 


2.40 


10.18 


Atypical I 
Atypical I 
Atypical I 
II 


1 
3 
4 
1 




5.36 
6.54 
1.72 


7.66 
8.92 
5.99 
2.03 




12.65 
1.64 

2.05 
0.62 


9.12 
8.61 
7.87 
6.54 


II 


2 


Trace 


1.86 


2.83 




0.08 


6.75 


II 


3 




1.75 


3.20 




2.09 


9.68 


III 


1 




4.58 


6.11 




3.10 


7.75 


III 


2 




2.56 


3.75 




1.08 


9.99 


III 


3 


Trace 


2.06 


3.41 




1.76 


9.95 



* Elek's modification of Pregl's micro method, 
f Pregl micro-Kjeldahl method. 
X Van Slyke method. 



By precipitating a concentrated neutral solution of the Type I 
cellular substance with hydrochloric acid, Wadsworth and Brown, 
just as Heidelberger, Goebel, and Avery had done, obtained an 
acid-insoluble and an acid-soluble fraction. 

When tested with homologous antipneumococcic serum, both of 
the fractions in a dilution of 1 to 6,000,000 gave precipitation. 
When a Type I rabbit serum, which had been absorbed separately 
with each fraction, was tested with the other, the acid-soluble frac- 
tion failed to precipitate serum absorbed by either, while the acid- 
insoluble fraction precipitated to a slight degree with the serum 
absorbed with the acid-soluble portion. Unfortunately, no protec- 
tion tests were carried out with the immune serum absorbed with 
these fractions, so it is impossible to make a close comparison of 
the results with those of Enders. 

Small doses of the Type I cellular carbohydrate were effective, 
by intraperitoneal injection, in protecting mice against a fatal 



CHEMICAL CONSTITUENTS 273 

dose of culture administered seven days after the immunizing dose. 
No data were given as to the degree of immunity established or as 
to its duration. 

The cellular carbohydrate caused an acute inflammatory proc- 
ess in the lung of the rabbit when introduced into the trachea. It 
also induced specific anaphylactic shock in sensitized guinea pigs. 
The cellular carbohydrate therefore agreed in its properties and 
activities somewhat closely with the A substance of Enders and 
appeared to be a substance apart from the soluble specific sub- 
stance of Heidelberger and Avery, and from the C substance of 
Tillett and Francis. 

COMPARISON OF VARIOUS CELLULAR CARBOHYDRATES 

For the purpose of comparing the properties of the various cel- 
lular carbohydrates derived from Pneumococcus, Wadsworth, 
Crowe, and Smith 1469 studied the absorption spectra of prepara- 
tions at different stages of purification. The preparations studied 
were those which had been used in chemical and immunological 
studies previously reported from their laboratory and, in addition, 
a specifically reacting substance from an atypical strain of Pneu- 
mococcus, originally derived from a virulent Type I standard cul- 
ture, together with a Type I soluble specific substance from the 
virulent culture. The absorption curves of solutions of the soluble 
specific substance of a virulent and of an attenuated Type I strain 
showed no significant differences. The curve of one preparation of 
a Type I cellular carbohydrate approximated that for the soluble 
specific substance, but that for another preparation of a Type I 
cellular carbohydrate approached the curve of the atypical strain 
of Pneumococcus. 

The authors pointed out that there was considerable evidence in 
the literature that a large number of carbohydrates show only con- 
tinuous absorption in the ultra-violet region of the spectrum but, 
when contaminated even slightly, yield marked absorption bands. 
Their work led to a contrary conclusion, since it was found that 



274 BIOLOGY OF PNEUMOCOCCUS 

the selective absorption in the ultra-violet region was character- 
istic of the pure substance. In conclusion, Wadsworth, Crowe, and 
Smith stated, however, that the curves suggested that since their 
preparations were not in a pure state, the more striking differences 
in the curves might be attributable to substances not concerned in 
serological or antigenic activity or in the production of purpura. 

TYPE VIII CARBOHYDRATE 

Brown 157 has published a description of a preparation of the 
Type VIII carbohydrate isolated by the methods employed by 
Wadsworth and Brown. She found that the soluble specific sub- 
stance was best removed from the broth concentrates of Pneumo- 
coccus by precipitation as the barium or calcium salt and by re- 
peated alcoholic precipitation. On analysis one preparation gave a 
nitrogen content of 0.19 per cent, phosphorus 0.06 per cent, ash 
0.70 per cent, with 3.90 per cent moisture. The specific rotation 
was about +126°, and while the substance before hydrolysis failed 
to reduce Fehling's solution, after boiling for four hours with 10 
per cent sulfuric acid, it yielded 69.5 per cent of reducing sugars 
calculated as dextrose. 

This VIII carbohydrate in a dilution of 1 to 4,000,000 gave a 
precipitate with Type VIII antiserum, and in a dilution of 1 to 
2,000,000 with Type III serum. When Type VIII serum was ab- 
sorbed with Type III soluble specific substance, it still precipitated 
with Type VIII carbohydrate in the same dilution as before ab- 
sorption ; but, when the same serum was absorbed with the homolo- 
gous carbohydrate, it failed to precipitate with either. Also, Type 
III SSS removed from Type III antiserum the precipitins for both 
Types III and VIII polysaccharides, but Type VIII carbohy- 
drate removed only the homologous precipitins. 

This new carbohydrate produced purpura in mice but its action 
was partly neutralized by Types III and VIII antipneumococcic 
rabbit serum and was intensified bv two similar antiserums from 



CHEMICAL CONSTITUENTS 275 

the horse. The Type VIII polysaccharide also induced fatal 
anaphylactic shock in guinea pigs passively sensitized with the ho- 
mologous antiserum from the rabbit but not with a similar serum 
obtained from the horse. 

Among the many methods applied to the purification of pneumo- 
coccal carbohydrate, ultrafiltration was employed by Brown. 158 
By passing broth cultures of Type VIII Pneumococcus through a 
Sharpies centrifuge and then filtering the clarified effluent through 
parlodion filters, or by passing broth cultures through a single 
nitrocellulose-coated alumina thimble, the author effected the 
elimination of inert material with a consequent enhancement of 
serological activity. 

ISOLATION OF AN UNIDENTIFIED CONSTITUENT OF THE PNEUMO- 
COCCAL, CELL 

In the interval elapsing between the first and third papers of 
Wadsworth and Brown, Felton 413 reported the isolation of what he 
claimed to be yet another constituent of the pneumococcal cell. 
This derivative was described as a non-carbohydrate and probably 
a non-protein substance, possessing the ability to immunize mice 
against pneumococcal infection. Supplementing his preliminary 
announcement, Felton 417 in October, 1934, published information 
which, to a considerable extent, clarified the doubt concerning the 
true nature of his antigenic "non-poly saccharide and probably 
non-protein derivative" of Pneumococcus. In the earlier paper, 
Felton reported that this crystalline substance gave a negative 
biuret test, a negative Molisch reaction, and also failed to cause 
precipitation in antipneumococcic horse serum. Further study, 
however, showed "that the crystalline material lacked immunizing 
properties, and that in all likelihood the crystals were leucine. In 
addition, although the substance gave negative Molisch reaction, it 
was found that the difference in the reduction of copper before and 
after hydrolysis corresponded to from 0.25 to 0.5 per cent glu- 



276 BIOLOGY OF PNEUMOCOCCUS 

cose. In other words there is present in this fraction a complex 
carbohydrate."* 

Felton 417 amplified the study of the acid-soluble and acid-insolu- 
ble fractions of Pneumococcus on which he had already published 
a preliminary note. 416 The material was prepared by dehydrating 
broth-grown pneumococci with acetone, followed by dessication in 
vacuo over calcium chloride. Prepared by this method the organ- 
isms retained their antigenicity at least through the period of the 
study. Watery suspensions of the dried pneumococci were treated 
first with enough sodium hydroxide to make the concentration of 
the alkali one-tenth normal, and then after the suspensions were 
allowed to stand at room temperature for one-half hour, an equal 
volume of various mineral and organic acids was added. In this way 
it was possible, as Heidelberger and Avery, and Wadsworth and 
Brown had found, to separate the pneumococcal material into an 
acid-soluble and an acid-insoluble fraction. The former possessed 
most of the immunizing activity of the cell, and the immunity pro- 
duced by injecting this fraction into white mice proved to be 
.largely type-specific. The latter fraction, probably containing 
some intact cells, also possessed a small amount of the immunizing 
substance and evoked in mice a heterologous immunity. Precipita- 
tion of the acid-soluble fraction with ethyl alcohol or acetone 
yielded at least 90 per cent of the immunizing substance. 

Felton's experiments, therefore, confirmed the work of Schie- 
mann and Caspar, 1228 Saito and Ulrich, 1214 Wadsworth and 
Brown, 1466 and Zozaya and Clark 1590 in that active immunity can 
be produced in white mice by a fraction smaller than the intact 
pneumococcal cell. The precipitating action of the acid-insoluble 

* In another part of the same paper, Felton stated that in his experience the 
Molisch test failed in the presence of other organic substances to indicate the 
presence of polysaccharide which on hydrolysis gave a glucose content of from 
0.2 to 0.5 per cent, and emphasized the fact that the biuret test for protein is 
notably insensitive, giving a positive test with concentrations of the majority of 
proteins in a dilution no higher than 1 to 10,000. When one considers the ex- 
traordinary immunological activity of bacterial polysaccharides and proteins, it 
need scarcely be said that less reliance should be placed on these or similar 
tests as criteria for determining the precise chemical nature of these antigens. 



CHEMICAL CONSTITUENTS 277 

fraction was also similar to that of the C Fraction of Tillett and 
Francis, but Felton ventured no closer comparison of the im- 
munizing principle in the acid-soluble fraction with the soluble spe- 
cific substance of Heidelberger and Avery. 

Looking back on the descriptions of the properties of the origi- 
nal soluble specific substance of Heidelberger and Avery, this ar- 
ray of diverse pneumococcal carbohydrates was confusing. One 
wondered which, if any. of these several substances obtained from 
Pneumococcus actually represented the specific polysaccharide as 
it existed preformed in the bacterial cell, or which one most closely 
approached the native substance in its chemical and antigenic fea- 
tures. There was the possibility, of course, that Pneumococcus 
might contain more than one constituent of this general type, with 
differences in individual composition which would account for their 
special immunological properties. 

CAUSE OF DIFFERENCES IN CARBOHYDRATES ISOLATED FROM 
PNEUMOCOCCUS 

In the brief communication by Pappenheimer and Enders 1049 
published in October, 1933, there was a definite clue to the cause of 
chemical and antigenic differences in some of the various carbohy- 
drates isolated from Pneumococcus. Enders had previously ob- 
served that the immunological activity of the A substance was rap- 
idly destroyed by heating on the alkaline side of neutrality, and so 
Pappenheimer and Enders surmised that in the method of Heidel- 
berger and Avery for the preparation of soluble specific substance 
the A substance was destroyed at the stage where it was precipi- 
tated with barium hydroxide.* Pappenheimer and Enders, in pre- 
paring the specific polysaccharide, using the simplified method of 
Heidelberger and Kendall, 620 accordingly took the precaution of 
maintaining an acid reaction throughout the process. In this way 
there was obtained from Type I Pneumococcus an extremely hy- 

* Dudley and Smiths*o also noted that heating with alkali destroyed the pre- 
cipitating activity of a preparation of pneumococcal polysaccharide made by 
their method. 



278 BIOLOGY OF PNEUMOCOCCUS 

groscopic, white, amorphous powder, soluble in the range pH 1 to 
9, and containing no sulfur or phosphorus. Its elementary analysis, 
amino nitrogen content, and specific rotation were practically 
identical with the soluble specific substance as prepared by Heidel- 
berger, Goebel, and Avery. 

The soundness of the reasoning was shown by the outcome of 
precipitin tests with this new preparation of the A substance and 
SSS. Both, in a dilution of 1 to 4,000,000, precipitated Type I 
immune serum. When the serum was absorbed with soluble specific 
substance, whereas SSS no longer gave a precipitate, the A sub- 
stance produced a precipitate with the SSS-absorbed serum even 
when added in a concentration of 1 to 4,000,000. On the other 
hand, the A substance completely removed the precipitin for both 
itself and the soluble specific substance. The authors had, by obvi- 
ating the injurious effect of alkali on the carbohydrate, succeeded 
in largely preserving the chemical and antigenic integrity of the 
specific polysaccharide of Type I Pneumococcus, and had supplied 
an explanation for one of the basic causes of the varied immuno- 
logical behavior of the different carbohydrate preparations previ- 
ously reported. 

RELATION OF THE ACETYL GROUP TO THE IMMUNOLOGICAL AC- 
TIVITY OF PNEUMOCOCCUS 

The diversity of the several polysaccharides had also naturally 
perplexed Avery and Goebel who, quite independently of Pappen- 
heimer and Enders and while their wprk was in progress,* set 
about the acquisition of a fuller knowledge of the nature of the 
relationship existing between the specifically reacting derivatives 
studied by other investigators and the type-specific polysaccharide 
formerly described by Avery and his colleagues. In a communica- 
tion published in 1933 Avery and Goebel 46 presented evidence that 
they had isolated the soluble specific substance in a chemical form 

* The paper by Pappenheimer and Enders appeared in October, 1933, and 
that by Avery and Goebel in December of the same year. 



CHEMICAL CONSTITUENTS 279 

more closely approximating that in which it probably exists as a 
natural constituent of the cell capsule. They identified the type- 
specific carbohydrate present in the intact bacterial cells and in 
filtrates of autolyzed broth cultures as an acetyl polysaccharide. 
According to Avery and Goebel this naturally occurring acetyl 
polysaccharide differs chemically from the specific carbohydrate 
as originally isolated principally in respect to the presence of ace- 
tyl groups, which endow the native substance with additional spe- 
cific properties not possessed by the polysaccharide after the re- 
moval of these labile groups by alkaline hydrolysis. Avery and 
Goebel claimed that, owing to the marked instability of the acetyl 
groups and the ease with which they are removed by treatment 
with alkali, the soluble specific substance as originally isolated, al- 
though still retaining the dominant type-specificity of the native 
substance, had, through the loss of its acetyl groups, suffered a 
corresponding loss of certain specific properties possessed only by 
the acetyl polysaccharide itself. Avery and Goebel further stated 
that the specific differences between the properties of the cell frac- 
tions studied by other investigators and those of the soluble spe- 
cific substance as originally defined, appeared, as a result of their 
experiments, to be due to the presence or absence of acetyl groups 
in the polysaccharide molecule. They wrote : "Indeed, so distinctive 
are the immunological reactions of the acetyl polysaccharide and 
those of the deacetylated derivative, that it is now possible to 
clarify many of the apparently conflicting views still current con- 
cerning the nature and properties of the specific carbohydrate of 
Pneumococcus Type I." 

By modified methods, in which treatment with alkali was pur- 
posely avoided, the soluble specific substance of Pneumococcus 
Type I was isolated by Avery and Goebel in the form of an ash- 
free, acetyl polysaccharide possessing marked acidic properties. It 
was readily soluble in water, and gave solutions of high viscosity 
which showed a specific optical rotation of about +270°. The 
naturally acetylated Type I polysaccharide was found to contain 



280 BIOLOGY OF PNEUMOCOCCUS 

4.85 per cent of nitrogen, approximately one-half of which was 
liberated in the amino form when the substance was treated with 
nitrous acid in the cold. It did not reduce Fehling's solution until 
after hydrolysis with dilute mineral acids. At the same time that 
reducing sugars appeared in the solution, the serological speci- 
ficity of the acetyl polysaccharide was destroyed. In this respect 
its behavior was identical with that of the deacetylated polysac- 
charide. 

The acetylated carbohydrate evidently contained uronic acids. 
It was soluble in water and in 80 per cent acetic acid. Aqueous 
solutions were precipitated by phosphotungstic acid, silver nitrate, 
and neutral and basic lead acetate, and were incompletely precipi- 
tated by barium hydroxide. Unlike the deacetylated product, the 
acetyl polysaccharide was precipitated by tannic acid but not by 
uranyl nitrate. It gave no color reaction with iodine-potassium 
iodide solution. It did not immediately decolorize weak solutions of 
potassium permanganate, and it gave negative reactions to the 
biuret, ninhydrin, sulfosalicylic, and picric acid tests. No traces of 
phosphorus or sulfur were detectable in the most highly purified 
preparations of the specific acetyl polysaccharide. 

The table on page 281, copied from the article by Avery and 
Goebel, shows the results of the analyses of the acetyl polysaccha- 
ride of Pneumococcus Type I. 

The analytical data presented in the table show that the de- 
acetylated product (Preparation 2 A), obtained by alkaline hy- 
drolysis of the acetyl polysaccharide (Preparation 2), contained 
no acetyl groups and was in all respects chemically identical with 
the polysaccharide that had hitherto been known as the soluble 
specific substance. This result agrees with that of Heidelberger and 
Kendall 617 who previously had found that the Type I polysaccha- 
ride (deacetylated) contained no acetyl groups. 

The results of the analysis corresponded closely with the calcu- 
lated composition of the acetylated polysaccharide, and this sub- 
stance, therefore, approached in its chemical make-up that of the 



CHEMICAL CONSTITUENTS 



281 



ANALYSES OF THE ACETYL POLYSACCHARIDE OF PNEUMOCOCCUS 

TYPE I 







a 


a 
o 








B 


c 






pi? 


Highest 








s 










0) 









si 


dilution of 


Preparation 


Source 


'3 








o 


1* 




3 


x"0 

bc>; 


polysacch- 
aride reacting 


No. 




!2 




.e 






s 

1 




c 
6 


V 


— 

a 
o 




with anti- 
pneumococ- 
cus serum 






< 


C/3 


< 


U 


X 


H 


< 


< 


S- 


0J 












Per 


Per 


Per 


Per 


Per 


Per 


Per 


Per 












cent 


cent 


cent 


cent 


cent 


cent 


cent 


cent 




1 


Bacterial 
cells 


— 


+270° 


0.0 


— 


— 


4.89 


2.30 


5.9 


0.0 


— 


1:5,000,000* 


2 


" " 


— 


+265° 


0.0 


— 


— 


4.86 


2.21 


6.9 


— 


— 


1:5,000,000* 


3 


Autolyzed 
broth 
cultures 


576 


+277° 


0.0 


42.55 


6.58 


4.85 


2.22 


6.0 


0.0 


32.0 


1:5,000,000* 


2 A, deace- 


t 


535 


+297° 


0.0 


+ 


+ 


5.05 


2.50 


0.0 


— 


27.6 


1 :5,000,000§ 


tylated 



























* Type I antipneumococcus serum previously absorbed with Preparation 2 A 
(deacetylated). 

t This sample of deacetylated polysaccharide was obtained by alkaline hy- 
drolysis of Preparation 2. This material is identical with the carbohydrate 
formerly known as the soluble specific substance of Type I Pneumococcus. 

t An analysis of carbon and hydrogen was made on a sample of deacetylated 
carbohydrate which had been reprecipitated five times at its isoelectric point. 
The material contained no ash, and had a carbon content of 40.33 per cent and 
a hydrogen content of 6.23 per cent. 

§ Unabsorbed Type I antipneumococcus serum. 



soluble specific substance as it probably exists in the pneumococ- 
cal cell. This hypothesis is further strengthened by the antigenic 
behavior of the acetylated polysaccharide when compared with 
that of the intact pneumococcus and with that of the soluble spe- 
cific substance as it is released from the cell during natural autoly- 
sis. The experimental evidence presented by Avery and Goebel goes 
a long way toward reconciling the conflicting differences in some of 
the various carbohydrate derivatives isolated by other workers 
from the pneumococcal cell and its products. 

The same authors found that both the acetyl polysaccharide and 
the deacetylated polysaccharide were precipitated by homologous 
immune serum in the highest dilution tested, representing a final 
concentration of one part in three million. When, however, the se- 



282 BIOLOGY OF PNEUMOCOCCUS 

rum was absorbed with the deacetylated polysaccharide, after re- 
moval of all precipitins for this form of the specific carbohydrate, 
the serum still reacted with the acetyl polysaccharide in equally 
high dilution. On the other hand, after absorption with the acetyl 
polysaccharide, the serum was completely exhausted of all precipi- 
tins for both forms of the carbohydrate, as shown by the absence 
of reaction when tested with each substance in dilutions ranging 
from 1 to 20,000 to 1 to 3,000,000. The deacetylated polysaccha- 
ride, therefore, selectively removed from the serum only the pre- 
cipitins for itself, whereas the acetyl polysaccharide completely 
removed all the precipitating antibodies for both forms of the spe- 
cific substance. 

Avery and Goebel discussed these results by saying: 

The specific precipitation of the acetyl polysaccharide in serum pre- 
viously absorbed with the deacetylated carbohydrate, and the readiness 
with which the former substance is converted into the latter by heat, are 
similar to the relationships observed by Enders, and by Wadsworth and 
Brown, between the substances isolated by them and the soluble specific 
substance which they prepared according to methods previously de- 
scribed in this laboratory. Since the specific substance thus prepared is 
now known to be the deacetylated polysaccharide, it seems not improb- 
able that the differences they observed, like those noted in Table II, 
represent the reactions not of two different carbohydrates but of a sin- 
gle substance in two chemically different forms ; namely, the naturally 
acetylated and the artificially deacetylated polysaccharide. 

Avery and Goebel also found that after absorbing Type I anti- 
pneumococcic serum separately with acetyl and deacetylated Type 
I polysaccharides, the serum absorbed with the former carbohy- 
drate no longer agglutinated Type I pneumococci, while immune 
serum treated with the latter substance still contained specific ag- 
glutinins. The authors next tested the ability of the acetyl and the 
deacetylated polysaccharides to absorb the protective antibodies 
from specific immune serum and discovered that while the deacety- 
lated carbohydrate reduced the titer of protective antibodies, it 
failed to remove them all, since the serum, absorbed by this sub- 



CHEMICAL CONSTITUENTS 283 

stance, according to the conditions of the test, in dilutions of 1 to 
10 to 1 to 100 still protected mice against virulent pneumococcal 
infection. On the contrary, the acetyl polysaccharide, under the 
experimental conditions employed, exhausted the serum of protec- 
tive antibodies. 

Further evidence of the specific antigenicity of the acetyl poly- 
saccharide as compared to the incomplete antigenic action of its 
deacetylated derivative was furnished by Avery and Goebel's ex- 
periments on the immunizing action of the two carbohydrates on 
mice. Three injections of 0.5 cubic centimeters each of a 1 to 
2,000,000 solution of the acetyl polysaccharide protected all the 
mice tested six days later against 10" 5 cubic centimeters of a cul- 
ture of Type I Pneumococcus, of which 10" 8 cubic centimeters 
killed the control mice. A similar series of injections of the same 
amounts of the deacetylated polysaccharide, however, failed to af- 
ford any protection to the mice tested. 

The outcome of the experiments just cited would explain the 
previous consistent failure of Avery and his associates to induce 
active immunity in mice and rabbits with their former prepara- 
tions of the soluble specific substance, which were then used only 
in the deacetylated form. "This difference in antigenic action, like 
that already noted in the serological behavior of the two forms of 
the polysaccharide, is referable to known differences in chemical 
constitution."* 

A careful examination of the chemical and immunological data 
presented in this communication would seem to justify Avery and 
Goebel's conclusion: 

An analysis of the specific reactions of the acetyl polysaccharide dis- 
closes a previously unsuspected similarity between this form of the spe- 
cific carbohydrate and the antigenically active fractions described by 
other investigators. From the chemical and immunological properties of 
the acetyl polysaccharide it seems highly probable that this substance in 
the purified state accounts for the antigenic action of the carbohydrate 

* Avery and Goebel. 



284 BIOLOGY OF PNEUMOCOCCUS 

of Schiemann and Caspar and of Wadsworth and Brown. As in the case 
of these substances, the acetyl polysaccharide is antigenically effective 
in mice only when administered in extremely minute quantities. Al- 
though an extensive study of the purpura-producing action of the acetyl 
polysaccharide has not been made, in several instances purpura has 
been noted in mice injected with amounts of this substance ranging 
from 0.4 to 4.0 mg. . . . That the antigenic action of the water-soluble 
fraction of Perlzweig and his co-workers may have been due to the 
presence of traces of unhydrolyzed acetyl polysaccharide seems not un- 
likely from the readiness with which it lost its immunizing capacity 
when heated in alkaline solution. 

The correspondence of the acetyl polysaccharide to the A sub- 
stance of Enders and to the cellular carbohydrate of Wadsworth 
and Brown has already been mentioned. The exact relation of the 
acetyl polysaccharide to the "non-carbohydrate and probably non- 
protein" derivative of Pneumococcus described by Felton still re- 
mains to be determined. 

Avery and Goebel had found, under the conditions of their ex- 
periment, that the acetylated polysaccharide failed to induce any 
immune response in rabbits. The serum of the treated animals 
contained no demonstrable antibodies, and the animals were not 
protected against subsequent infection with organisms of the ho- 
mologous type. It was shown, furthermore, that the acetyl poly- 
saccharide persisted in the circulation of the treated rabbits for 
considerable periods of time, was slowly excreted by the kidney, 
and appeared in the urine in its naturally acetylated form. This 
observation, particularly when compared to the immunizing action 
of the same substance in mice, has an important bearing on the 
definition of antigenicity. 

In 1934, Goebel, Babers, and Avery 523 sought a better under- 
standing of the immunological significance of the acetyl group in 
these complex pneumococcal polysaccharides. For this purpose the 
authors synthesized the p-aminophenol (3-glucoside of glucose and 
its monoacetyl ester and then combined these two glucosides with 
horse serum globulin by means of the diazo reaction. These syn- 



CHEMICAL CONSTITUENTS 285 

thetic carbohydrate-azoproteins were employed as antigens in the 
production of immune rabbit serum. From the experiments, involv- 
ing homologous precipitation and specific inhibition of precipita- 
tion, confirmation was obtained of the view previously expressed by 
Avery and Goebel that the immunological specificity of carbohy- 
drates is determined by their stereochemical configuration, and 
their data lent support to the further assumption that the intro- 
duction of a simple chemical group, such as the acetyl radical, en- 
dows a carbohydrate with a new and distinct specificity which is 
determined by the chemical nature of the group thus introduced. 
The differences exhibited by these two purely synthetic carbohy- 
drate azoproteins accurately paralleled the differences in the sero- 
logical specificity exhibited by the acetylated and deacetylated 
polysaccharides of Type I Pneumococcus. 

Additional information concerning the A substance of Enders 
was presented in the 1934 communication of Enders and Wu, 362 
who prepared the A substance according to the procedure given by 
Pappenheimer and Enders and the soluble specific substance by the 
method of Heidelberger, Goebel, and Avery. The immune serum was 
obtained by the repeated intravenous injection of rabbits with Type 
I pneumococci in 0.3 per cent formalinized saline solution, and also 
of suspensions of pneumococci killed by heating at 56° and 60°. 
The immunological properties of the two polysaccharides were 
then tested by the bactericidal method of Ward and by mouse-pro- 
tection tests in which the mice had been treated with antipneumo- 
coccic rabbit serum and also by active immunization with the A 
substance. 

After the completion of the study, Enders and Wu announced 
that the A substance possessed greater anti-opsonic action than 
either the deacetylated carbohydrate obtained by boiling in alkali 
or the SSS of Type I Pneumococcus prepared according to the 
method of Heidelberger, Goebel, and Avery. The A substance 
practically eliminated the opsonic titer of normal human serum — 
an effect not observed with equivalent amounts of the deacetylated 



286 BIOLOGY OF PNEUMOCOCCUS 

material or the soluble specific substance — while in immune serum 
the A substance brought about a quantitatively greater reduction 
in opsonic activity than its derivatives, although the authors were 
not able to demonstrate complete inhibition of phagocytic action 
by the method of absorption of antibody. The A substance by ab- 
sorption lowered the mouse-protective titer of Type I antipneumo- 
coccic rabbit serum to a greater degree than did a similar treat- 
ment with the deacetylated carbohydrate. Analogous to the acetyl 
polysaccharide of Goebel and Avery, the A substance, adminis- 
tered in very small quantities, protected mice against an otherwise 
fatal dose of Type I Pneumococcus, although doses larger than 
0.005 milligrams failed to establish protection in the animals. This 
particular antigenic action of the A substance was impaired by 
boiling in 0.02N sodium hydroxide, and was destroyed by similar 
treatment with 0.1N sodium hydroxide. 

Enders and Wu also determined that after the injection of the 
A substance into mice active immunity arose within three days fol- 
lowing the injection, reached its height in from six to twenty-five 
days thereafter, and became retrogressive by the forty-ninth day 
following vaccination. As might be expected, the injection of the A 
carbohydrate into immunized mice immediately before giving an 
infective inoculation abolished the active immunity, while the serum 
of mice actively immunized with the A substance conferred passive 
immunity on normal mice. The authors suggested that, since the 
evidence which had accrued in the course of their study indicated 
that the A carbohydrate obtained from Type I Pneumococcus and 
the acetyl polysaccharide of Avery and Goebel represented the 
same chemical substance, the designation A carbohydrate or A 
substance be relinquished in favor of the more accurately descrip- 
tive term, acetyl polysaccharide. 

Although Enders and Wu did not include the cellular carbohy- 
drate of Wadsworth and Brown in their comparison, taking into 
consideration their observations together with those of Aver}' and 
Goebel, there is good reason to believe that some of the prepara- 



CHEMICAL CONSTITUENTS 287 

tions of cellular carbohydrates, though presumably in impure 
form, are practically identical with the A substance and the acetyl 
polysaccharide. There is, nevertheless, one point of difference for 
which as yet no explanation has been forthcoming, and that is the 
presence of sulfur and phosphorus reported by Wadsworth and 
Brown in their preparations. Heidelberger never found sulfur or 
phosphorus in the specific polysaccharides of Type I, II, and III 
pneumococci and since Avery and Goebel have stated that their 
highly purified preparation contained neither of these elements, it 
becomes difficult to reconcile these differences. 

The work of Enders and Wu, taken with that of Avery and 
Goebel, would seem to demonstrate that the A substance and the 
soluble specific substance approached very closely in antigenic 
function the hypothetical specific polysaccharide as it exists in the 
intact pneumococcal cell. There developed in the latter's study one 
point, however, which raises a doubt as to the exact common iden- 
tity of the isolated specific carbohydrate and the native capsular 
polysaccharide of Pneumococcus. The fact that the former, by ab- 
sorption, failed to remove all the protective antibodies from the 
homologous immune serum might be taken to mean that in its isola- 
tion it had suffered the loss of some completing molecular group, 
or possibly that its full antigenic power is exerted only when it is 
in combination with some other constituent of the pneumococcal 
cell. 

Another striking difference between the acetyl and the deacety- 
lated polysaccharides of Pneumococcus was manifested in the be- 
havior of these two derivatives toward the blood-group specific 
substance A. Witebsky, Neter, and Sobotka 1526 in 1935 announced 
that a relationship between the soluble specific substance of pneu- 
mococci and the blood-group substance A of man could be demon- 
strated by the inhibition of sheep-cell hemolysis by a group-specific 
A-antiserum, although the various types exhibited certain quanti- 
tative differences. When, however, the deacetylated carbohydrate 
was used, it failed to react with the group-specific A-antiserum, 



288 BIOLOGY OF PNEUMOCOCCUS 

while the acetyl polysaccharide, under the conditions of the test, 
exerted an inhibitory influence on sheep-cell hemolysis by the 
A-antiserum up to a dilution of 1 to 1,000,000 of a one per cent 
solution. Furthermore, the authors could demonstrate the activity 
of the acetyl polysaccharide by complement fixation and by inhibi- 
tion of group-specific iso-agglutination. 

Witebsky, Neter, and Sobotka reported another interesting ob- 
servation on the properties of the acetyl polysaccharide. When 
this substance was treated with the feces filtrate previously de- 
scribed by Schiff and Akune, 1233 by Schiff and Weiler, 1234 later by 
Witebsky and Satoh, 1527 and still more recently by Sievers, 1285 it 
lost much of its inhibitory action toward the group-specific A-anti- 
serum, and also its ability to inhibit the iso-agglutination of Group 
A blood cells. Moreover, the acetyl polysaccharide of Type I Pneu- 
mococcus, after having lost its reactivity toward the group-spe- 
cific A-antiserum following treatment with feces filtrate, still re- 
acted with Type I antipneumococcic serum that had previously 
been absorbed with deacetylated Type I polysaccharide. 

Witebsky, Neter, and Sobotka, 1526 in the introduction to their 
communication, mentioned the correlation between the Forssman 
antigen and the blood-group specific substance of human blood- 
group A. This fact, considered along with the relation existing be- 
tween pneumococci and the blood-group specific substances A and 
B as reported by Bailey and Shorb, 66 and the known carbohydrate 
nature of the blood-group substance A as demonstrated by Land- 
steiner, 780 Landsteiner and Levene, 781 and by Brahn, Schiff and 
Weinmann, 146 and then the isolation by Freudenberg and Eichel 482 
from the urine of men belonging to group A of a carbohydrate 
closely resembling in its chemical structure the type-specific poly- 
saccharide of Pneumococcus of Avery, Heidelberger, and Goebel, 
reveals a field of investigation which yet remains to be explored. 

RECENT METHODS OF PREPARING CAPSULAR POLYSACCHARIDE 

The effect of alkali in impairing or destroying the antigenicity 
of the specific polysaccharides of Pneumococcus was tested by Fel- 



CHEMICAL CONSTITUENTS 289 

ton (1934), 416 who reported that a Type I immunizing antigen, 
when heated at 100° in 0.1N sodium hydroxide lost 50 per cent of 
its antigenic strength after one hour of heating, and 94 per cent in 
two hours. With a Type II antigen in 0.5N sodium hydroxide, the 
immunizing activity remained the same as that of the control after 
four hours' heating. The same sample of Type I antigen heated in 
0.1N acetic acid for two hours lost 50 per cent and in 0.1N hydro- 
chloric acid 87 per cent of its immunizing power. Its precipitating 
action, however, remained the same. Of the samples of SSS pre- 
pared by the technique of Avery and Goebel and heated under the 
same condition, Type I in one test showed destruction of antigen- 
icity and, in a second, no loss ; with Type II no loss was observed. 
In a miscellaneous group of pneumococcal fractions with immuniz- 
ing activity in dilutions of 1 to 50,000,000 to 1 to 2,000,000, and 
with variation of hydrolyzable sugar from 12 to 0.25 per cent, ab- 
sorption end-point from 8 to 0.1 per cent, and precipitin titer from 
1 to 5,000,000 to 1 to 5,000, Felton found that there was no defi- 
nite correlation between acetyl group content and immunizing ac- 
tivity. However, acetyl groups were found in the samples studied 
ranging from 6.4 per cent in one sample which immunized in a dilu- 
tion of 1 to 5,000,000, to 1.2 per cent in a sample which immunized 
in the same dilution. Inasmuch as this information was published 
merely in abstract form without the presentation of any experi- 
mental data, it is impossible to set a proper value upon its signifi- 
cance. 

In 1935, Felton, with Kauffmann and Stahl, 430 gave the details 
of a method that had been employed in their laboratory for five 
years in the routine preparation of soluble specific substance of 
pneumococci. It had been planned to eliminate the time-consuming 
initial evaporation of the broth culture and to minimize any altera- 
tion of the chemical and antigenic structure of the carbohydrates. 
Calcium phosphate was employed as a selective adsorbent for sepa- 
rating the polysaccharide from the culture medium and its con- 
stituents. 

Felton and his co-workers stated that the product from the first 



290 BIOLOGY OF PNEUMOCOCCUS 

had shown characters differing from those of the soluble specific 
substance of Heidelberger and Avery. The new preparation pre- 
cipitated more protein from a given immune serum ; it consistently 
removed all the protective antibody from an homologous serum; 
and it produced immunity in white mice. The authors were not in- 
clined to grant the validity of Avery and Goebel's explanation that 
this immunizing property depended upon the presence of the acetyl 
group in the polysaccharide. They had, it seems, in a comparative 
test succeeded in actively immunizing white mice not only with 
their preparations but also with a sample of soluble specific sub- 
stance prepared by Heidelberger by his original method. Some 
statement as to the presence or absence of the acetyl group in their 
and Heidelberger's own preparation would have been helpful in ar- 
riving at a just appraisal of their contention. 

In the latest paper to come from Felton's laboratory (1936), 
its authors (Felton and Prescott) 431 present evidence which, in 
their opinion, challenges the validity of the theory that the specific 
antigenic properties of pneumococcal polysaccharides are due to 
the presence of the acetyl group in the molecule. Because of the 
seeming heterodoxy of the claim, the summary and conclusions are 
here repeated practically verbatim : 

A method has been indicated by which the linkage between the units 
in the polysaccharide of Type I pneumococcus are altered with con- 
current changes in biological activity. This alteration is shown by both 
the high titer in bisulfite and iodine reactions in the original material 
and the absence in samples B* and C* of reducing sugars on hydrolysis 
after destruction of the aldehyde groups in hot NaOH solution. 

* Sample A was a purified preparation consisting of a mixture of seven sam- 
ples of Type I polysaccharide prepared by different methods both from the 
supernatant broth (of pneumococcal cultures) and from the bacterial cell 
( Pneumococcus ) . 

Sample B was the original A dissolved at pH 7 in a concentration of 1 mg. 
per cc, and then made alkaline to N/10 concentration with NaOH and heated 
in an Arnold sterilizer at 100°C. for 30 minutes. To the alkaline solution after 
cooling were added 2 volumes of a 1 : 1 alcohol-ether mixture. The white precipi- 
tate which formed was washed thoroughly with alcohol, alcohol-ether, and dried. 

Sample C was made from the foregoing fraction (B) by treating a neutral 
aqueous solution, containing 1 mg. per cc, with one-tenth of the volume of con- 
centrated NH 4 OH at 4°C. for 18 hours. The NH 4 OH solution was precipitated 



CHEMICAL CONSTITUENTS 291 

From this altered SSS, four samples were prepared by various alka- 
line treatments and tested both chemically and biologically in compari- 
son with the original material. Each of the samples may be considered 
separately. The original material (A) gives tests for aldehyde groups, 
is high in "acetyl" content, and is optically as well as immunologically 
active. In B, a portion of A treated for 30 minutes in N/10 NaOH at 
100°C, the optical activity is lost, the aldehyde groups are removed, 
"acetyl" content and all immunological activities are greatly reduced. 
In C, sample B treated with NH 4 OH, there is a reduction of acid from 
vacuum distillation, no indication of sugar on acid hydrolysis, but an 
increase of the iodine reaction. Optical rotation is changed from zero to 
-}-190 o , or higher than the control. Immunologically also this sample is 
more active than the original material (A). Sample D is the original 
material (A) treated with NH 4 OH. There is a decrease of 90% in acid 
on vacuum distillation, a slight decrease in glucose number, a higher 
optical rotation than the original material, as well as a high titer in all 
immunological tests. It is significant that with a 90% decrease in "ace- 
tyl" content the immunizing activity is higher than the original. There 
is also a significant increase in its combining equivalent with antibody. 
Sample E is the NH 4 OH treated control (D) further treated with hot 
NaOH as in sample B. The NH 4 OH treatment caused a rearrangement 
of the molecule or stabilization, for on acid hydrolysis glucose number 
was 10.25 as compared to zero with sample B. Optical activity was re- 
duced but not entirely lost. Immunological tests, although decreased, 
were not as low as in sample B, with the exception of the combining 
equivalent and that was the lowest of all samples, 0.9 unit. The acid 
from vacuum distillation was higher than in sample D, indicating a 
splitting off of the terminal carboxyl radicals. 

At this stage of our investigation certain inferences may be made: 
(1) An antigen has been prepared from a Type I polysaccharide which 
in our opinion is non-protein in nature for the following reasons: (a) 
treatment with hot NaOH (N/lO) and then NH 4 OH results in a prod- 
uct more highly antigenic than the original preparation, and (b) all 
well-recognized protein tests are negative including a test for sulfur 
with a large sample of material. (2) This antigen may be considered 

by dilution with 2 volumes of 1 : 1 alcohol-ether, washed repeatedly with alcohol, 
alcohol-ether and dried. 

Sample D was made from the original material (A) by treatment with 
NH.OH as in C. 

Sample E was prepared from D by the same procedure as B, in other words 
heating at 100°C. in the Arnold sterilizer in N/10 NaOH for 30 minutes. 



292 BIOLOGY OF PNEUMOCOCCUS 

non-polysaccharide since both the Molisch test and the test for reduc- 
ing sugars after acid hydrolysis are negative. (3) The antigens so pre- 
pared from Type I SSS produce active immunity in mice against both 
Type I and Type II pneumococci. (4) The restoration of the biological 
properties destroyed with hot NaOH by treatment with NH 4 OH shows 
that the "acetyl" content is of no significance in determining the bio- 
logical activity of the preparation studied, but conversely indicates that 
this property is determined by a definite molecular configuration which 
is readily altered by strong alkalis. 

Believing that the procedures hitherto employed in the prepara- 
tion of pneumococcal polysaccharides might have disrupted the 
molecular configuration of the carbohydrate molecule with a con- 
sequent loss of essential radicals, Sevag (1934) 1257 applied gentler 
measures for their isolation. Adopting the well-known action of 
liquid air in disintegrating bacterial cells, Sevag first froze the 
sediment from twelve-hour dextrose-serum broth cultures of a viru- 
lent strain of Type I Pneumococcus and then subjected the detri- 
tus to prolonged shaking in a mixture of water, chloroform, and 
amyl alcohol. The protein coagulated by this treatment was re- 
moved and the polysaccharide in the supernatant fluid was iso- 
lated by precipitation with alcohol. After further purification with 
chloroform, an alcohol-insoluble fraction was obtained which was 
claimed to be protein-free, gave a strong Molisch reaction, and 
contained between 6.60 and 6.72 per cent nitrogen, of which from 
1.21 to 1.42 per cent was in the form of amino nitrogen. The spe- 
cific rotation of the product was [a] D = + 218.3* to 219.1* 
which after acid hydrolysis became +54.1 and showed a glucose 
content of 23.98 per cent. The polysaccharide thus isolated, in a 
dose of 0.0001 milligrams, protected mice against a thousand fatal 
doses of virulent Type I Pneumococcus and in high dilutions gave 
a high precipitation titer with homologous immune rabbit serum. 

Sevag entertained some doubts concerning the validity of Avery 
and Goebel's claim that the capsular polysaccharide of Pneumo- 
coccus Type I was an acetylated substance and queried whether 

•Typographical error in original figures gives +21.83 and 21.91. 



CHEMICAL CONSTITUENTS 293 

the acetyl group might have been introduced into the carbohydrate 
molecule by the repeated treatment of the substance with acetic 
acid. Sevag accordingly excluded acetic acid from the method of 
preparing a special sample and found, contrary to expectations, 
that the polysaccharide, on analysis, had an acetyl content of 7.7 
to 7.97 per cent as against the figure of 2.57 per cent obtained by 
Avery and Goebel. 

It might reasonably be argued, therefore, that Sevag, by a less 
harsh treatment of pneumococcal cellular material, had succeeded 
in isolating a polysaccharide that more closely represented the 
carbohydrate as it naturally exists in the cell than did the prepa- 
ration of Avery and Goebel. Apparent confirmation of the assump- 
tion came subsequently with the experimental results of Heidel- 
berger, Kendall, and Scherp (1936). 627 In searching for a reason 
for the differences in the immunological behavior of acetylated and 
deacetylated polysaccharides and from the alkali-treated carbohy- 
drates of Type II and Type III pneumococci, the authors devised 
a method of preparation in which the use of heat, strong acid, or 
alkali was avoided. In general the procedure consisted of the con- 
centration of culture filtrate in vacuo to a convenient volume; 
separation of the polysaccharide from salts and protein degrada- 
tion products by repeated precipitation with alcohol in the presence 
of sodium acetate and acetic acid; removal of proteins by dena- 
turation with chloroform and butyl instead of amyl alcohol, as 
used by Sevag ; and elimination of any glycogen or starch present 
by methods depending upon the properties of the individual poly- 
saccharides. The products were isolated as the neutral sodium salts. 

The analytical data on the polysaccharides obtained by the re- 
vised method present many points of interest when compared with 
results of analyses of older preparations. In the case of Type I 
polysaccharide the new product contained 4.62 per cent nitrogen 
with 2.0 per cent in the amino form, against 5.12 and 2.5 per cent 
for the old ; showed specific rotation of +278 against +305, the 
acetyl content was 7.1 as compared to 3.4; it had a higher vis- 



294 BIOLOGY OF PNEUMOCOCCUS 

cosity, and precipitated more antibody nitrogen from homologous 
antiserum from both horse and rabbit than did the earlier prod- 
ucts. When compared with the substance isolated by Sevag, the 
high nitrogen, low amino nitrogen, and optical rotation indicated 
to Heidelberger and his associates the presence of a nitrogen-con- 
taining component not present in their products, but there was at 
the time no way of determining whether the component was an im- 
purity or an integral part of the polysaccharide as it exists in the 
organism. 

The study of Heidelberger, Kendall, and Scherp also revealed 
that, contrary to earlier opinion, the specific polysaccharides are 
not thermostable. On heating preparations from pneumococci of 
Types II and III there was an accompanying and marked drop in 
viscosity without any change in reactivity with homologous anti- 
serum. There was a decrease in precipitating power in the case of 
Type I polysaccharide owing to a partial removal of acetyl. The 
authors suggested that unheated preparations have the largest 
particle size or longest chain, and that heating results in a degra- 
dation of the molecule to smaller units. 

In the same communication the authors reported the actual iso- 
lation of the methyl glucoside of galacturonic methyl ester from 
the products of hydrolysis of the Type I polysaccharide by methyl 
alcoholic hydrochloric acid. 

From the properties of the new preparations, Heidelberger, Ken- 
dall, and Scherp believed that while the products might be arti- 
facts just as were the older ones, they were certainly a step closer 
to the native substances themselves and designated the substances 
as specific polysaccharides of Types I, II, and III Pneumococcus 
with the abbreviations SI, SII, and SIII.* 

* For the purpose of obtaining a maximal yield of pneumococci with a high 
polysaccharide content, O'Meara and Browniosi devised a medium consisting of 
peptone, glucose, sodium chloride and sodium bicarbonate, potassium phosphate, 
and thioglycollic acid in water. In this medium, Type I Pneumococcus grows 
rapidly and abundantly. The organisms are found to be rich in capsular poly- 
saccharide, whereas the medium contains a minimal amount of free polysac- 



CHEMICAL CONSTITUENTS 295 

The susceptibility of the capsular polysaccharide to even fairly 
rigorous chemical treatment has been shown further in the case of 
the soluble specific substance of Type III Pneumococcus by Hor- 
nus and Enders. 656 By avoiding as far as possible the use of strong 
acids in the isolation of the carbohydrate a preparation was ob- 
tained that gave a precipitate with Type III immune serum after 
the serum had been absorbed by Type III SSS made by the earlier 
method of Heidelberger and Avery. The sample contained 0.3 per 
cent nitrogen and in this respect resembled the material described 
by Heidelberger, Kendall, and Scherp, 626 but it differed in some of 
its serological properties. 

The gap between the polysaccharides as derived by chemical ma- 
nipulation and the native substance in the pneumococcal cell is be- 
ing still farther narrowed. By omitting the preliminary autoclav- 
ing of pneumococcal cultures, by leaving out alkaline treatment, 
and employing a method that minimized hydrolysis by acid or 
alkali, Chow 225 obtained a polysaccharide from Type I Pneumo- 
coccus that gave a precipitate with homologous immune rabbit se- 
rum previously absorbed with the acetyl polysaccharide. The ace- 
tyl polysaccharide failed to react with homologous immune rabbit 
serum after absorption by the new carbohydrate. The greater ab- 
sorptive power of the new preparation as compared with that of 
the acetyl polysaccharide suggests its possession of a group or 
radical which was lacking in the acetylated derivative, but the fact 
that antipneumococcic serum absorbed with the new preparation 
was still agglutinative and specifically protected white mice against 
an otherwise fatal dose of Type I Pneumococcus may be taken to 
indicate that the carbohydrate isolated by Chow is not so com- 

charide. The details of the preparation of the medium are given in the Ap- 
pendix. 

The use of peptone instead of meat as recommended by O'Meara and Brown, 
according to an unpublished personal communication of Heidelberger, greatly 
facilitates the preparation of both the capsular polysaccharide and the C car- 
bohydrate. The C Fraction can be easily isolated from the supernatant fluid of 
such a broth after removal of the capsular polysaccharide. 



296 BIOLOGY OF PNEUMOCOCCUS 

plete in its antigenic or chemical constitution as the hypothetical 
polysaccharide existing preformed in the pneumococcal cell. While 
Chow's statement may be true that his polysaccharide may be the 
parent substance from which the acetyl polysaccharide could be 
obtained by appropriate treatment, there is little doubt that fur- 
ther search must be made in the lineage of pneumococcal carbohy- 
drate for the original progenitor of the many derivatives which 
have been described. 

OTHER PHYSICOCHEMICAL PROPERTIES OF THE CAPSULAR POLY- 
SACCHARIDE 

Heidelberger and Kendall 618 studied other physicochemical prop- 
erties of the specific polysaccharides of pneumococci, and deter- 
mined the viscosity, conductance, and behavior in diffusion of the 
sodium salts of Type III and other capsular polysaccharides. The 
high values of the equivalent conductance indicated that the so- 
dium salt of Type III S* is a strong electrolyte characterized by a 
mobile negative ion of very high valence. Under varying conditions 
of salt concentration the authors determined the viscosities of I, 
II, and III S and correlated the findings with the magnitude of the 
charge on the anion. The experimental data indicated that at least 
eight or ten carboxyl groups were present in the molecule at regu- 
lar intervals of every 340 of molecular weight, so the cumulative 
effect of the negative charges on the Type III S would be very 
large, which should result in large interionic or Coulomb forces. 
These forces were found to be strong. 

The specific polysaccharide of Type I Pneumococcus showed 
viscosity abnormalities of a similar character, but of smaller mag- 
nitude. Although its acid equivalent was even lower than that of 
Type III S, Heidelberger and Kendall thought that internal com- 
pensation of negative charges by the basic groups present was the 
cause of the smaller effect. Further data on the specific gum arabic 

* The substitution of the letter S for SSS is consistent with Heidelberger's 
original usage. 



CHEMICAL CONSTITUENTS 297 

and Type III Pneumococcus polysaccharide showed that the 
viscosity effects decrease with the relative number of carboxyl 
groups, or negative charges in the molecule. 

SUMMARY 

It should by no means be assumed that the last word has been 
written about pneumococcal polysaccharides. Notwithstanding the 
fact that the presence in any appreciable percentage of what is 
considered the acetyl group may exert a marked influence on the 
antigenic properties of these substances, we should not close our 
minds to the many possibilities which the subject presents. We 
cannot as yet be sure that any of the substances isolated from 
Pneumococcus are truly representative of the actual components 
of the living cell, even though the immunological action of the ace- 
tyl polysaccharide most nearly approaches that of the unbroken 
Pneumococcus. 

We can be sure that many of the methods employed up to the 
present time have led to products far from pure by any standard 
we set, and we can be equally certain that the majority of the pro- 
cedures have caused more or less disturbance of the molecular ar- 
rangement of the cells' constituents with a consequent loss of 
chemical and immunological integrity. Many of the preparations 
hitherto derived from Pneumococcus by natural means must be 
looked upon as mixtures of carbohydrate, protein, and other cellu- 
lar elements, and the majority of the preparations produced by 
chemical methods cannot be accepted as anything but artifacts 
containing, to be sure, a nucleus of intrinsic antigenic material 
but, nevertheless, representing substances which eventually may be 
discovered to be removed in varying degree from the native antigen. 

There are chemical groups other than the acetyl radical in pneu- 
mococcal capsular polysaccharides, and until we have a more thor- 
ough analysis and more exact identification of all these substances 
supported by their respective immunological reactions, it would be 
folly to take any dogmatic stand in this highly complex question. 



298 BIOLOGY OF PNEUMOCOCCUS 

A careful examination of the specifications which have been 
given in all these reports would enable one to make a tentative and 
partial chemical reconstruction of the pneumococcal cell. The 
basic protoplasm would contain protein — a nucleoprotein — with a 
more or less constant composition for all pneumococci regardless 
of serological type. This common component would, in some re- 
spects, bear a close resemblance to the protein of other members 
of the genus, Streptococcaceae, and a more general resemblance to 
that of other more distantly related microorganisms. Another con- 
stituent of the protoplasm would be lipids, probably combined with 
the protein and conceivably loosely linked to the carbohydrate. 
Then the substance of the cell would also include a polysaccharide 
— a carbohydrate of the nature of the C Fraction of Tillett and 
Francis. This phosphorus and nitrogen containing carbohydrate 
would, like the nucleoprotein, be possessed by every Pneumococcus, 
whether of full virulence or degraded below the stage of any type 
identity. There would naturally be inorganic salts, but since little 
is known about them, their actual composition cannot be defined. 
In this viable, watery solution of colloids and crystalloids there 
would be enzymes — proteases, lipases, invertases, and saccharases 
— active in the metabolic processes of the cell and potentially able 
to destroy it. 

This globular mass of protoplasm would be surrounded by a 
shielding envelope or capsule, largely carbohydrate in nature. The 
capsular material would be present when the cell was living in a fa- 
vorable environment, in greatest amount when the surroundings 
were ideal, but entirely lacking when the cell had suffered from se- 
vere degenerative processes. But with or without it, these particu- 
lar bacterial cells would still be pneumococci, their identity as such 
resting upon their somatic nucleoprotein and carbohydrate. 

The normal Pneumococcus, however, would possess in its capsule 
a polysaccharide that would serve to distinguish the organism 
from many of the other members of the species. These complex car- 
bohydrates would differ in their content of nitrogen, of phos- 



CHEMICAL CONSTITUENTS 299 

phorus, and possibly of other elements, some being well supplied 
with one or the other of these elements, while others would have 
none. The carbon, hydrogen, and oxygen would be combined to 
form varying percentages of carboxyl, acetyl, or other radicals, 
and the presence and proportion of these groups, together with the 
varying sugars chemically combined, would serve to identify the 
special serological type to which the pneumococcus belonged. The 
capsular polysaccharides, furthermore, would differ in their acidic 
and basic affinities and in their relation to the plane of polarized 
light. The proportion of these carbohydrates would fluctuate in 
the different types, and on hydrolysis the substances would yield 
unequal amounts and various kinds of cleavage products such as 
glucosamines, sugar acids, and the simpler sugars. With the com- 
mon protein and the somatic carbohydrate as a nucleus, it would 
be feasible, as soon as other specific polysaccharides are isolated, 
to hypothesize a basic reconstruction of all the thirty-two and pos- 
sibly other as yet undiscovered serological types of pneumococci. 

The capsular polysaccharides would be linked to some of the 
protein, or even to the lipids, in a combination susceptible to cleav- 
age by the enzymes of the cell, to the disruptive effects of physical 
forces, to the lytic action of bile, or to the hydrolyzing action of 
chemical agents. Their chemical structure is frail, and to the chem- 
ist they present difficulties in the way of isolating them as they 
exist in their native state. They call for the utmost care in their 
separation lest their molecular configuration be distorted or muti- 
lated. 

While the somatic constituents are sufficient to insure the exist- 
ence of the cell under ordinary circumstances, the capsular struc- 
ture composed of type-specific polysaccharides is essential to the 
complete functioning of the cell as a pathogenic organism, and it is 
the latter specific complex that gives to a pneumococcus type- 
specificity and its individual place in the biological and immuno- 
logical scheme. The capsular material conditions and is associated 
with the difference between a state of saprophytism and parasit- 



300 BIOLOGY OF PNEUMOCOCCUS 

ism, and endows the cell with its highly specialized powers for 
stimulating the tissues of the animal body to the elaboration of 
substances antagonistic to the type-specific polysaccharide and to 
the cell as a whole. 

The immunological properties of Pneumococcus and of its chemi- 
cal constituents and derivatives will be discussed in more detail in 
later chapters. 



CHAPTER IX 

SPECIFIC POLYSACCHARIDE-SPLITTING 
ENZYMES 

The decomposing action of bacterial enzymes on the soluble spe- 
cific substance of pneumococci, with a description of the micro- 
organisms, of the physical and biochemical properties of the en- 
zymes and their effect on pneumococci in the test tube and in 
experimentally infected animals. 

The discovery of the specific polysaccharides of Pneumococcus 
was an incentive to a search for enzymes capable of decom- 
posing these complex carbohydrates. During pneumococcal infec- 
tions in man the soluble specific substance is present in the blood 
and since it is excreted unchanged by the kidneys it was reason- 
able to expect that none of the body tissues would be able to at- 
tack this substance. No tissue has ever been found to possess the 
power. Avery and Dubos 42 were the first to make a systematic hunt 
for a ferment possessing the ability to digest the capsular poly- 
saccharide of Pneumococcus. The authors tested enzymes from 
animal and plant sources known to be active in the hydrolysis of 
simpler carbohydrates, but all failed to affect the polysaccharides. 
The similarity of these bacterial carbohydrates to hemi-cellulose 
led the authors to search among the molds, yeasts, actinomyces, 
and bacteria known to decompose substances allied to the cellu- 
loses, but here again the quest was fruitless. 

FIRST ISOLATION OF POLYSACCHARIDE-SPLITTING ENZYME : SIII 
BACILLUS 

Avery and Dubos reasoned that localities where large amounts 
of organic materials — especially materials belonging to the group 
of hemi-celluloses — accumulate and undergo decomposition were 
most likely to harbor the desired organism. So they tried leaf mold, 



302 BIOLOGY OF PNEUMOCOCCUS 

composts of corn-cob, rye straw, sphagnum, oak leaves, farm ma- 
nure, and soils rich in organic matter, such as peat soils and soils 
heavily manured, and finally came to examine a sample of soil from 
a cranberry bog. It seemed a far cry from a pneumococcus grow- 
ing in the lung of man and, among its other vital activities, build- 
ing up a complex carbohydrate, to a lowly saprophytic bacillus 
capable of decomposing the specific capsular polysaccharide 
formed by Pneumococcus. Such, nevertheless, was the case. 

In 1930, Avery and Dubos announced that a mixed bacterial 
suspension made from bog soil was able to split the specific capsu- 
lar polysaccharide of Type III Pneumococcus, and from the mot- 
ley crowd of bacteria in this peat, they succeeded in isolating a 
pleomorphic, motile, spore-bearing bacillus that was responsible 
for the breaking down of the Type III polysaccharide. 

In their next communication, Avery and Dubos 43 gave fuller de- 
tails of the isolation, cultivation, vital characters, and enzymatic 
action of this curious bacillus. The authors mentioned the earlier 
observation of Toenniessen, 1413 who had found that when Bacillus 
vulgatus was seeded together with encapsulated Friedlander ba- 
cilli, the latter organisms grew deprived of their capsule, and then 
went on to describe the isolation of the new organism. The mineral 
medium used was based on one previously described by Dubos 329 
for the isolation of cellulose-decomposing bacteria, with the addi- 
tion of the capsular polysaccharide of Type III Pneumococcus in 
final concentrations varying from 0.001 to 0.2 per cent. The pneu- 
mococcal polysaccharide was the only source of organic carbon in 
the medium. 

This mineral medium was selected because it contained no nu- 
trient substances that would act as readily available sources of 
energy, so that the bacillus, when deprived of any other food, 
would attack the specific pneumococcal polysaccharide — an exam- 
ple of the so-called "starvation" phenomenon. By repeated trans- 
plantation and dilution, and by heating the inoculum at 70°, Dubos 



POLYSACCHARIDE-SPLITTING ENZYMES 303 

and Avery finally discovered the spore-bearing bacillus which they 
designated as the "SIII bacillus." 

When grown in the synthetic medium, the organism appears as a 
minute Gram-negative bacillus, at times smaller than the Pfeiffer 
bacillus. It contains metachromatic granules ; it grows diffusely in 
peptone solution and in this medium sedimentation of the growth 
occurs after several days' incubation. When cultivated in peptone 
solution or in plain broth the organism appears as a fairly large, 
actively motile bacillus, having peritrichous flagellae, the young 
cells measuring 2 to 3|J by 0.5p. Short chains and diploforms are 
often observed. In broth the bacilli are at first Gram-positive and 
do not autolyze readily, but in the mineral medium the bacilli rap- 
idly become Gram-negative and undergo almost immediate self- 
digestion. The spores resist heating for thirty minutes at 75° but 
are killed by boiling for five minutes. On plain nutrient agar free 
of dextrose, growth occurs in the form of small, whitish colonies, 
two millimeters in diameter, circular, slightly raised, umbilicated, 
with entire edge and fairly smooth surface. 

In the media tested, the organism is strictly aerobic and exerts 
its saccharolytic action on Type III Pneumococcus within the 
range pH 6.2 to 7.8 at room temperature and at 37.5°. The iso- 
lated enzyme, however, is equally active under both anaerobic and 
aerobic conditions, and it is likely that this soluble principle be- 
longs to the group of hydrolytic enzymes. 

Dubos and Avery then found that the enzymatic action of the 
culture could be enhanced by frequent transplantation, and that 
the enzyme was present in filtered autolysates of the cultures. The 
action of the enzyme was limited, of all the substances tested, to 
the Type III polysaccharide, since it failed to decompose the car- 
bohydrates of Types I, II, and VIII* Pneumococcus, and those of 
Types A, B, and C of Friedlander's bacillus, of Hemophilus influ- 
enzae Type a, and of gum arabic which, it may be recalled, gives a 

* Personal communication from Dubos. 



304 BIOLOGY OF PNEUMOCOCCUS 

precipitin reaction with Type III antiserum. The activity of the 
enzyme was destroyed by heating for ten minutes at 60° to 65°. 
Neither normal beef nor rabbit serum had any inhibiting effect on 
its activity. 

As a means for measuring the enzymatic strength, Dubos and 
Avery determined the minimal amount of a given enzyme prepara- 
tion which would decompose one cubic centimeter of a 0.001 per 
cent solution of specific Type III capsular polysaccharide in 
eighteen hours at 37.5°, the decomposition of the carbohydrate be- 
ing demonstrated by the disappearance of precipitating action in 
the presence of Type III antipneumococcic serum. As the authors 
stated, "The specific decomposition of the capsular polysaccharide 
of Type III Pneumococcus, by the organism as well as by the en- 
zymes it produces, illustrates once more the specificity of the types 
of Pneumococcus, and confirms the fact that the capsular poly- 
saccharides, and not some impurities carried along with them, are 
responsible for type-specificity." 

EFFECT OF THE ENZYME ON THE CELE CAPSULE 

Inasmuch as the capsular substance in its native state forms a 
morphological structure which conditions the antigenic and sero- 
logical reactions of the pneumococcal cell as a whole as well as its 
power to invade and multiply in the animal body, it became of spe- 
cial interest to ascertain what effect this specific enzyme would 
have on the encapsulated cells growing in vitro and in vivo. To this 
end, therefore, Avery and Dubos performed a series of experi- 
ments the results of which revealed the fact that the enzyme by it- 
self is neither bacteriostatic, bactericidal, nor bacteriolytic, and 
that, without impairing the viability of the cocci or without inhib- 
iting the ability of the cells to synthesize polysaccharide, the en- 
zyme, by decomposing the specific carbohydrate, merely deprives 
the bacteria of their capsules. It was evident that the action of the 
enzyme did not destroy the function of elaborating the capsular 
substance, since organisms decapsulated by enzyme again formed 




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Photomicrographs by Louis Schmidt After Avery and Dubos* 

ACTION OF ENZYME AGAINST TYPE III PNEUMOCOCCUS 



POLYSACCHARIDE-SPLITTING ENZYMES 305 

specific polysaccharide and regained their capsule when trans- 
ferred to a medium free of the enzyme. 

The action of the enzyme against Type III Pneumococcus is 
shown in the accompanying photomicrographs, a description of 
which follows : 

1. A stained preparation of the peritoneal exudate of a mouse two 
hours after the intraperitoneal injection of 0.01 cubic centimeter of a 
virulent culture of Type III Pneumococcus. The bacteria show well- 
defined capsules, and no evidence of phagocytosis is seen. Many poly- 
morphonuclear and a moderate number of mononuclear leucocytes are 
present. (Gram stain. X 1000.) 

2. A corresponding preparation of the exudate of a mouse two hours 
after receiving the same amount of culture together with 0.5 cubic centi- 
meter of a preparation of the specific enzyme. The bacteria are devoid 
of capsules. Polymorphonuclear leucocytes predominate and phagocyto- 
sis is evident. (Gram stain. X 1000.) 

3. A stained film of the peritoneal exudate of a mouse four hours 
after injection with 0.01 cubic centimeter of culture alone. The bac- 
teria are increased in number, encapsulated, and extracellular. The cel- 
lular elements are polymorphonuclear and mononuclear leucocytes in 
about equal numbers. (Gram stain. X 1000.) 

4. A corresponding preparation of the exudate of a mouse four hours 
after receiving the same amount of culture together with 0.5 cubic centi- 
meter of a preparation of the specific enzyme. Marked phagocytosis 
has occurred and only an occasional organism is seen outside the ac- 
cumulated leucocytes, nearly all of which are of the polymorphonuclear 
type. (Gram stain. X 1000.) 

PROTECTIVE ACTION OF ENZYME IN MICE 

Avery and Dubos then studied the protective action of the en- 
zyme in mice against Pneumococcus Type III infection. By vary- 
ing the amount of enzyme with a constant amount of culture, by 
keeping constant the dose of enzyme and by decreasing the quan- 
tity of culture, by giving the enzyme before and simultaneously 
with the infecting dose of culture, and by administering the en- 
zyme by a single injection into mice eighteen hours after the onset 
of infection, the authors demonstrated that under the conditions 



306 BIOLOGY OF PNEUMOCOCCUS 

of the experiments, 0.1 cubic centimeter of the enzyme afforded 
mice protection against one million times the fatal dose of virulent 
Type III Pneumococcus. Avery and Dubos also showed that again 
the action of the enzyme was specific for Type III; that the 
greater the activity of the enzyme in vitro the greater was its pro- 
tective action in mice ; and that the enzyme exerted a curative ac- 
tion since the mice receiving the enzyme eighteen hours after the 
onset of infection recovered. The extraordinary enzymatic princi- 
ple, therefore, not only strips the capsule from Type III Pneumo- 
coccus, but renders innocuous a large multiple of the infecting dose 
of the organism and, what is more, under certain conditions it ac- 
tually cures mice of an already active infection. 

PHILOSOPHICAL ASPECT OF THE ACTION OF THE ENZYME 

The possibilities presented by this and other similar enzymes in 
the prevention and cure of pneumococcal infection in man were 
strikingly promising, but before going on to experiments leading in 
that direction, it may be interesting and more profitable to con- 
sider first the important philosophical aspects of the action of an 
enzyme of this particular type on bacterial polysaccharides. It 
would be impossible to improve on Avery and Dubos' discussion of 
this phase of the subject, so it is quoted in full. 

The present study emphasizes the importance of the capsule in the 
biological reactions of the pneumococcus. It is, indeed, a significant 
fact, that no matter whether one regards this organism from the view- 
point of type-specificity, antigenicity, or its capacity to undergo varia- 
tion, or whether, as in the present instance, one considers the pneumo- 
coccus with reference to its virulence and fate in the animal body, the 
one dominant factor influencing all these phenomena is the function of 
the cell to elaborate the specific capsular polysaccharide. These rela- 
tionships, however, are not to be interpreted as meaning that virulence 
is dependent merely upon differences in the structural morphology of 
the bacterial cell. For it is a common observation that an encapsulated 
strain of Pneumococcus may be virulent for one species and not for an- 
other. However, it is equally true that the function of elaborating the 



POLYSACCHARIDE-SPLITTING ENZYMES 307 

specific capsular polysaccharide is most highly developed in pneumo- 
cocci that are best adapted to growth in the animal body. From this 
point of view, virulence and capsule formation, although not causally 
related, are at least intimately associated. When the function of forming 
the capsular substance is suppressed or inhibited, as in the case of the 
R. variants, or when, as in the present instance, although this function 
is unimpaired the capsule itself is destroyed by an enzyme, the naked 
bacteria are thereby exposed directly to attack by the phagocytes of the 
host. 

In this sense, the action of the enzyme may be said to result in pre- 
paring the encapsulated bacteria for phagocytosis ; not as in the case of 
antibodies, by specific sensitization, but by the process of decapsulation. 
In the former instance, the reaction is an immunological one, whereby 
the capsular material is altered by union with the type-specific anti- 
body ; in the latter case, the reaction is a chemical one in which the cap- 
sular polysaccharide is actually decomposed by the enzyme. Although 
the mode of action of both these specific agents is different in each in- 
stance, the end result, so far as the fate of the microorganism is con- 
cerned, is the same in both cases. 

It is of interest that although neither the enzyme nor the specific 
antibody is by itself bactericidal or bacteriolytic, yet each by reacting 
specifically with the capsular substance exposes the virulent organisms 
to the phagocytic action of the body tissues. The enzyme, like the spe- 
cific antibody, serves merely to initiate the protective reaction, the com- 
pletion of which is ultimately dependent for its successful issue upon 
the effective cellular response of the host. 

The present study also suggests that the capsule — long recognized as 
a defense mechanism on the part of virulent bacteria — is a decisive fac- 
tor in determining the fate of pneumococci in the animal body, and that 
this structure is vulnerable to attack by specific agents other than anti- 
bodies. 

METHODS OF PRODUCTION 

Dubos 335 " 6 then attempted the development of practical methods 
of production, purification, and concentration of the enzyme, and 
investigated the influence of certain factors on the potency and 
primary toxicity of the preparations. He first studied the growth 
and enzyme production of the SHI bacillus in more than fifty dif- 



308 BIOLOGY OF PNEUMOCOCCUS 

ferent media containing simple and complex saccharides, organic 
acids, alcohols, peptones, et cetera. Apart from the Type III spe- 
cific polysaccharide he found only one other substance which 
caused the elaboration of the specific enzyme by the SIII bacillus 
and that was the aldobionic acid derived from the capsular poly- 
saccharide of Type III Pneumococcus. When, however, he used an- 
other aldobionic acid, namely, that derived from gum arabic — a 
substance closely related immunologically to the Type III poly- 
saccharide^ — no growth and, therefore, no enzyme could be ob- 
tained in the synthetic medium. 

Dubos then learnt that the production of enzyme was also influ- 
enced by the concentration of capsular polysaccharide in the me- 
dium. The yield of enzyme rapidly increased with rising concentra- 
tions of the soluble specific substance, but decreased when the 
concentration exceeded 0.1 per cent and was inhibited by concen- 
trations of 0.3 per cent or higher. Yeast extract, along with the 
soluble specific substance, stimulated enzyme production, but Du- 
bos' first preparations in this medium proved to be toxic for ex- 
perimental animals. Yeast extract in itself was not toxic and only 
when acted upon by cultures did it become so. However, Dubos was 
able to reduce the toxicity of the enzyme preparation by lowering 
the concentration of yeast extract to 0.03 per cent, the minimum 
compatible with good yields of enzyme. 

Dubos then resumed his endeavors to produce a purer and more 
potent preparation of the polysaccharide-decomposing enzyme 
from the SIII bacillus, and in two communications (Dubos, 832 and 
Dubos and Bauer 338 ) reported the results. It was first found that 
the yield of enzyme was proportional within certain limits to the 
amount of inoculum ; that it increased with a rise in the concen- 
tration of capsular polysaccharide; and that 0.1 per cent of the 
carbohydrate represented the optimal amount for the purpose. 
After testing the influence of salt concentration in the medium on 
the metabolic activity of the SIII bacillus, on the basis of the col- 
lected data a method was devised which was described as follows 



POLYSACCHARIDE-SPLITTING ENZYMES 309 

The bacteria are grown in a solution of 2 per cent casein hydrolysate 
(pH 7.0) at 37 °C. and under conditions of strict aerobiosis; the cells 
from the 16 hour old culture, separated by centrifugalization, are re- 
suspended in small amounts of distilled water. 

A medium is prepared consisting of 0.1 per cent capsular polysaccha- 
ride and 0.1 per cent NaCl in distilled water. This medium is distrib- 
uted in 25 cc. amounts in large Erlenmeyer flasks (1 liter capacity) to 
provide for aerobic conditions, and each flask is inoculated with the cells 
recovered from 500 cc. of the culture of the SHI bacillus in the casein 
hydrolysate medium. The material is incubated for 12—18 hours at 
37°C. and the cultures tested to ascertain the disappearance of the spe- 
cific polysaccharide and the absence of contaminants. The cultures are 
now frozen and thawed repeatedly to secure the release of the endocel- 
lular enzyme. 

The enzyme is ultimately separated from the cell debris by filtration. 
However, since the cell suspension is very viscous, it is first subjected 
to the following treatment. The cell suspension is made alkaline to pH 
10.0 by the addition of sodium borate. Equimolecular concentrations of 
dibasic sodium phosphate and calcium chloride are then added to bring 
about a heavy precipitate of calcium phosphate which facilitates the 
clarification of the material by centrifugalization; (it had been estab- 
lished previously that the enzyme is not adsorbed on the calcium phos- 
phate at alkaline reaction). The supernatant which contains all the en- 
zyme in solution is now passed through a Seitz filter, then through a 
Berkefeld (V) filter. It is important to observe that the enzyme is com- 
pletely adsorbed on the asbestos pad of the Seitz filter; this, however, 
can be prevented by washing the filter with nutrient infusion-peptone 
broth previous to filtration. After this treatment, the enzyme passes 
through the filter without loss. The potency of this filtrate is such that 
0.002-0.004 cc. are required to decompose 0.01 mg. of the capsular 
polysaccharide under the conditions of the test. 

Dubos found that the SHI bacillus grew abundantly in casein- 
hydrolysate medium, but in this medium the organism did not form 
any appreciable amount of the enzyme responsible for the decom- 
position of the polysaccharide. When, however, large numbers of 
the bacilli grown in the casein medium were re-suspended in a solu- 
tion of the capsular polysaccharide which constituted the sole 
source of carbon, the specific carbohydrate substrate was rapidly 



310 BIOLOGY OF PNEUMOCOCCUS 

decomposed, and filtered autolysates of the cell suspension exhib- 
ited tremendously enhanced enzymatic activity. For a given 
amount of capsular polysaccharide decomposed in the new phos- 
phate-yeast medium, the yield of enzyme obtained was much larger 
than that recovered by the former cultural methods. The produc- 
tion of the enzyme always failed when conditions in the substrate 
were unfavorable to cellular multiplication. 

With Bauer, Dubos 338 then described an improved technique for 
concentrating and purifying the enzyme preparations. They fil- 
tered the solution, made in the manner just described, under fifty 
pounds pressure through collodion membranes of the type de- 
scribed by Bauer and Hughes. 90 The enzyme, under optimal condi- 
tions of filtration, passed through membranes of an average pore 
size of 10.6 mpi but was held back by pores having a diameter of 
8.2 mu. When the enzyme solutions were filtered to dryness through 
membranes of such porosity as to hold back the active principle, 
and when proper precautions were taken to prevent or minimize 
adsorption, such as the preliminary "greasing" of the collodion 
membrane with meat-infusion peptone broth, the enzyme could be 
completely recovered by immersing the membrane in distilled water 
or physiological salt solution. 

EFFECT OF ENZYME ON INFECTION INDUCED IN RABBITS 

In 1932, Goodner, Dubos, and Avery 536 studied the action of the 
enzyme on the dermal infection of rabbits induced with Type III 
Pneumococcus, employing the Type III strain virulent for rabbits 
previously described by Tillett. The culture was grown in rabbit- 
blood broth and its virulence was maintained for rabbits by fre- 
quent animal passage. Of the blood-broth culture 10" 8 cubic centi- 
meters given intraperitoneally sufficed to kill mice within ninety-six 
hours, and when given intradermally into rabbits 0.000,01 cubic 
centimeter caused death or a protracted disease of severe charac- 
ter. The enzyme preparations were, for the larger part, purified 
and concentrated by the method of Dubos. The intradermal infec- 



POLYSACCHARIDE-SPLITTING ENZYMES 311 

tions were induced by the procedure devised by Goodner and al- 
ready described in Chapter VI. 

The authors found that the injection of adequate amounts of 
the specific enzyme twenty-four hours after infective inoculation 
brought about an early and complete cessation of the disease. The 
blood stream was freed of pneumococci and the organisms disap- 
peared from the local lesion in the course of a few hours. Follow- 
ing the administration of the enzyme, the temperature at first rose, 
but fell within twenty-four hours to normal levels ; the local lesion 
failed to spread and soon showed signs of healing. The results ob- 
tained by Goodner, Dubos, and Avery in a large series of infected 
animals indicated that in cases with severe bacteriemia large quan- 
tities of the enzyme were necessary to obtain successful results, 
while in animals having fewer organisms in the blood at the time of 
treatment smaller amounts of the enzyme would suffice. 

The curative action of the enzyme was specific, at least in so far 
as it was tested. When given intravenously in a dose of one hun- 
dred units* twenty-four hours after inoculation with ten and one 
hundred minimal infective doses of Type I Pneumococcus, it failed 
to save the lives of the two rabbits tested. The authors found that 
the curative action of the enzyme was destroyed by heating at 70° 
for thirty minutes. 

In a subsequent communication appearing later in the same 
year, Goodner and Dubos 535 reported the results of studies on the 
quantitative action of the enzyme on the dermal infection of rab- 
bits with Type III Pneumococcus. With the same Type III culture 
and enzyme preparations similar to those employed in the previous 
experiments, using the number of pneumococci present in the blood 
as an indicator of the severity of the infection, Goodner and Dubos 
determined the amounts of enzyme required to bring about the re- 
covery of the animal. It was ascertained that the required dose of 
enzyme bore a definite relation to the number of organisms circu- 

* According to Dubos: "A unit of enzyme may be denned as one hundred 
times the smallest amount which will bring about the complete decomposition of 
0.01 milligrams of the purified specific capsular polysaccharide in 18 hours at 
37°C." 



312 BIOLOGY OF PNEUMOCOCCUS 

lating in the blood stream at the time of treatment. Thus, in ani- 
mals with negative blood cultures or with a low grade bacteriemia, 
an amount of enzyme as small as five units might suffice to save the 
life of the animal; with a bacteriemia of 100 to 1,000 organisms 
per cubic centimeter of blood, twenty units might be necessary; 
with a bacteriemia of 1,000 to 10,000 organisms fifty units were 
required. In rabbits in which the bacteriemia exceeded 10,000 or- 
ganisms per cubic centimeter a single injection of even one hundred 
units failed to rescue the animal, although infections of this order 
were successfully treated by repeated injections of large amounts 
of enzymes over a period of days. 

Goodner and Dubos, in the discussion of their experimental 
data, stated that, although the quantitative relation established in 
the experiments was between the amount of enzyme and the number 
of pneumococci present in the blood, the fundamental relation was 
that existing between the quantity of enzyme and the total amount 
of specific capsular polysaccharide present in the body ; and that 
an index of the latter was the degree of bacteriemia. The authors 
concluded that the enzyme is not a therapeutic agent per se, but 
one which, by decomposing the capsular substance of pneumococci 
and thus preparing the bacterial cells for phagocytosis — pianti- 
cating them, to use Friel's term — initiates a process which the 
body must be in condition to carry on if the animal is to recover. 
Hence, in the use of the enzyme, this capacity of the body must be 
reckoned with. 

EFFECT ON INFECTION INDUCED IN MONKEYS 

With this groundwork built, the next logical step was to apply 
the enzyme to the treatment of animals higher in the zoological 
scale which were suffering from experimental Type III Pneumococ- 
cus infection. Francis, Terrell, Dubos, and Avery, 477 accordingly, 
chose young adult monkeys — the Java monkey (Macacus cyno- 
molgos) — as test animals, and infected the animals with a strain 
of Type III Pneumococcus virulent for rabbits. They employed the 




Photographs by Louis Schmidt 



After Franc: 1 !, Terrell, Dubos, and Avery' 



LUNGS OF A MONKEY DURING THE COURSE OF EXPERIMENTAL 
PNEUMONIA TREATED THE FIRST DAY AFTER INFECTION 



POLYSACCHARIDE-SPLITTING ENZYMES 313 

technique developed by two of them (Francis and Terrell 476 ), who 
had found that by intratracheal or intrabronchial inoculation of 
Type III cultures it was possible to produce in monkeys of the 
Cynomolgos species an experimental pneumonia, which in its clini- 
cal aspects resembled pneumococcal lobar pneumonia in man. The 
authors used enzyme preparations made in the manner already de- 
scribed, which varied in potency from two to twenty units per 
cubic centimeter. They began the enzyme treatment within the first 
three days after inoculation, administering the enzyme prepara- 
tions intravenously usually in doses of ten cubic centimeters each, 
with additional treatments as often as three times daily, either in- 
travenously, intraperitoneally, or by both routes simultaneously. 
Francis, Terrell, Dubos, and Avery purposely made no attempt to 
ascertain the minimal amount of enzyme required in individual 
monkeys but, rather, they employed the treatment intensively until 
the result was assured. Moreover, they selected for enzyme treat- 
ment the sickest monkeys among those infected, since this plan 
would reduce the factor of natural recovery from the infection as 
well as put the enzyme to the severest test. 

The accompanying photographs show the effect of pneumonia 
on the lungs of a monkey that had been treated the first day after 
infection, as follows : 

1. Control. Before inoculation (December 19). 

2. Nineteen hours after infection and seven hours before the first 
treatment (December 20), showing well localized consolidation in lower 
half of the right upper lobe. 

3. Second day (December 21), showing extension of pneumonia 
throughout right upper lobe. 

4. Third day (December 22), showing increased density of the 
shadow over the right upper lobe, but no evidence of further spread. 

5. Fifth day (December 24). Resolution of the pneumonia has be- 
gun, as shown by the decrease in density and beginning aeration of the 
area. 

6. Tenth day (December 29), showing complete resolution of the 
pneumonic shadow. 



314 



BIOLOGY OF PNEUMOCOCCUS 



THE INFLUENCE OF ENZYME THERAPY UPON MORTALITY-RATE IN 

EXPERIMENTAL TYPE III PNEUMOCOCCUS PNEUMONIA 

IN MONKEYS* 





Untreated animals 


Treated animals 


Class of infection 


Total 


Recov- 
ered 


Died 


Per cent 
mortality 


Total 


Recov- 
ered 


Died 


Per cent 
mortality 


Pneumonia without 

Pneumonia with sep- 
ticemia (1-250)+.. 

Pneumonia with sep- 
ticemia (250- 
2000)+ 


20 
20 

12 
16 

68 

48 


20 
11 

3 



34 

U 



9 

9 
16 

34 

84 




45.01 

>56.2 
75.0 J 

100.0 

50.0 

70.8 


8 
15 

9 

8 
40 

32 


8 
15 

8 
1 

32 

24 





1 

7 
8 

8 



11.1 


Pneumonia with sep- 
ticemia (2000+) + 

Total 


J 
87.5 
20.0 


Total for groups 
•with septicemia 


25.0 



* Classified on the basis of height of septicemia in first three days. 
+ The numbers in parentheses in the left-hand column equal the cocci present in 
one cubic centimeter of blood. 



The condensed results of experiments on forty monkeys are 
given in the accompanying table. The figures in the table represent 
only the recovery or death of the monkeys, and do not give in de- 
tail the effect of the enzyme on the course of the pneumococcal in- 
fection. As Francis, Terrell, Dubos, and Avery reported, it can 
readily be seen that in the groups in which no invasion of the blood 
occurred, spontaneous recovery was to be uniformly expected, 
whereas in the extremely severe forms of the disease the great ma- 
jority of animals were too completely prostrated to respond to 
any therapeutic aids. Further comment of the authors on the ac- 
tion of the enzyme upon the infected monkeys can best be given in 
their own words : 

In addition to the apparently beneficial effects of specific enzyme 
therapy as measured by survival or death of the animals, certain other 
favorable influences were observed. In a high percentage of cases in 



POLYSACCHARIDE-SPLITTING ENZYMES 315 

which extension of the pneumonic process was occurring at the time of 
treatment, the spreading promptly ceased following the initial injection 
of enzyme. Although the density of the area of consolidation might at 
first appear greater than before treatment, extension did not occur and 
resolution of the lesion soon began. This limitation of spread of the 
pneumonia was not infrequently noted in the severe cases before the 
bacteria were completely eliminated from the blood stream. A compari- 
son of the ultimate degree of pulmonary involvement in the treated and 
untreated cases reveals the fact that it was less, in general, in the for- 
mer series. While in the treated cases the extension was apparently lim- 
ited early, in the untreated animals extension of the pneumonia pro- 
gressed, frequently with fatal results. 

That the administration of enzyme promoted sterilization of the 
blood stream seems certain. In the milder cases this occurred quite rap- 
idly. In animals in which the higher degrees of septicemia were present, 
there was rarely an increase, more regularly a prompt decrease in the 
number of pneumococci in the blood following the administration of en- 
zyme. Even in cases which eventually terminated fatally, or in which 
extreme septicemia occurred early in the disease, cultures of the blood 
showed a marked reduction in the number of bacteria within 4 to 5 
hours after the first treatment. 

Simultaneously with limitation of the pneumonia, beginning resolu- 
tion, and elimination of septicemia, a fall in temperature usually oc- 
curred. In fact, there was a tendency for the fever to subside concur- 
rently with the cessation of pneumonic spread, even though septicemia 
still persisted. Although a marked leukopenia was comparatively fre- 
quent at the time treatment was begun, the number of leukocytes rose 
with the beginning of recovery. 

In fatal untreated cases with septicemia, a high incidence of positive 
cultures was obtained from pleural or pericardial fluids at autopsy. In 
many instances frank empyema or pericarditis was present. In the 
treated cases with severe infections which resulted fatally, the incidence 
of these complications was also high. In recovered animals of the 
treated series, therefore, a frequency of suppurative complications equal 
to that of the untreated animals might be expected. The fact that the 
treated animals which survived recovered without suppurative sequelae 
suggests that enzyme therapy either prevented the development of em- 
pyema and pericarditis or was therapeutically effective even in the pres- 
ence of these complications. 

As previously stated, many technical difficulties have been encoun- 



316 BIOLOGY OF PNEUMOCOCCUS 

tered in attempting to produce enzyme preparations of uniformly high 
therapeutic activity and purity. The different lots of enzyme have, as a 
result, been inconstant in both these respects. In some instances toxic 
effects, attributable to impurities in the material, have been noted in 
animals after the administration of enzyme. These impurities may in- 
duce a febrile reaction and a decrease in the white blood count of the 
animal. At other times, when the animal is extremely ill with subnormal 
temperature and a marked leukopenia, the administration of impure 
preparations may produce a further depression of temperature and of 
the leukocytes. 

The results of the present study indicate that the specific enzyme, 
even in its present state of purity, exerts a favorable therapeutic effect 
upon the course and outcome of experimental Type III pneumococcus 
pneumonia in monkeys. Nevertheless, the present study again empha- 
sizes the therapeutic limitations of the enzyme. The action of the en- 
zyme is known to be exerted upon the capsular polysaccharide of Type 
III Pneumococcus. By being deprived of its capsule, the bacterium is 
made susceptible to phagocytosis by the cells of the animal body. How- 
ever, when the disease process is of extreme severity and the entire cel- 
lular mechanism of the body is markedly depressed, the animal may no 
longer possess the capacity to dispose of the organisms rendered vul- 
nerable by the specific action of the enzyme. 

ISOLATION AND STUDY OF OTHER POLYSACCHAREDE-SPLITTING 
ENZYMES 

In 1933, Sickles and Shaw 1281 discovered microorganisms, other 
than the SILT bacillus of Avery and Dubos, capable of decompos- 
ing the capsular polysaccharide of Type III Pneumococcus and, 
furthermore, succeeded in obtaining from soil another organism 
possessing an enzymatic action on the capsular polysaccharide of 
Type II Pneumococcus. From decaying vegetable matter from dif- 
ferent localities, Sickles and Shaw isolated in pure culture three 
strains of sporulating bacilli, which in morphology and some of 
their other characters differed from one another and from the SHI 
bacillus. The organisms were all aerobic, Gram-negative, motile 
rods with peritrichous flagellae, and formed oval spores wider than 
the vegetative cells. On beef-extract agar the organisms grew in 



POLYSACCHARIDE-SPLITTING ENZYMES 317 

colonies about two millimeters in diameter, which were yellowish 
white, smooth, and round with entire edges. On blood agar, two of 
the strains produced two types of colonies, one whitish and opaque 
and the other grayish and semi-translucent. One of the strains iso- 
lated from material from a decayed hickory stump covered with 
sphagnum moss appeared to utilize agar as well as the Type III 
polysaccharide. The cultures fermented dextrose, lactose, saccha- 
rose, maltose, dextrin, mannitol, xylose, galactose, inulin, and sali- 
cin as well as decomposing the specific polysaccharide of Type III 
Pneumococcus. 

Sickles and Shaw made their enzyme preparations in a mineral 
substrate, using the yeast extract medium of Dubos only for the 
seed culture. The cultures were usually incubated for three days at 
36°, filtered through a Berkefeld candle, and then concentrated by 
ultrafiltration through a 7.5 per cent pyroxyline membrane, the 
enzyme remaining on the membrane. In this manner a preparation 
was obtained with an enzymatic activity of approximately twenty 
units per cubic centimeter. The enzyme in a dose of 0.5 cubic centi- 
meters, when given eighteen hours after inoculation, protected mice 
against infection with ten minimal fatal doses of Type III Pneu- 
mococcus and, when given seven hours after inoculatidn, the ex- 
tract protected the animals against 1,000 fatal doses. 

The organism isolated by Sickles and Shaw from soil, possessing 
the power to decompose Type II soluble specific substance, dif- 
fered markedly from the bacillus attacking Type III SSS. It, too, 
was an obligate aerobe, but oval in form, resembling a yeast. It 
could be maintained by daily transfer on the mineral medium con- 
taining 0.01 per cent Type II soluble specific substance, but no 
growth could be induced in the mineral medium alone or on any of 
the usual nutrient solid media. No growth could be seen in meat- 
extract broth but microscopic examination indicated that slight 
multiplication had taken place. The organism grew slowly in meat- 
extract broth containing a fermentable carbohydrate, but did not 
grow in meat-infusion broth, litmus milk, or peptone water, on po- 



318 BIOLOGY OF PNEUMOCOCCUS 

tato slants, or in gelatine. Growth was similarly slow on a solid me- 
dium composed of agar and the mineral medium containing Type 
II polysaccharide. In the mineral medium to which one per cent of 
carbohydrate was added, some strains of the organism fermented 
maltose, xylose, and dextrin, while all strains decomposed lactose 
and saccharose. 

The cultures broke down the Type II cellular carbohydrate of 
Wadsworth and Brown as well as the soluble specific substance of 
the same type, but failed to dissolve the specific polysaccharides 
of Type I and III pneumococci. In the case of this organism, how- 
ever, the enzyme was active only when the body of the organism 
was present, but Sickles and Shaw apparently did not test filtrates 
from the cultures. At that time no experiments were performed on 
the protective properties of this culture. It was observed, how- 
ever, that the organism in decomposing the cellular carbohydrate 
robbed it of its purpura-producing property, but this effect may 
have been due to the digestion of admixed protein material of pneu- 
mococcal origin by a proteolytic ferment in the enzymatic prepa- 
ration employed. 

In a later paper, Sickles and Shaw 1283 reported the isolation 
from soil of another organism — a small Gram-negative bacillus — 
which acted on the Type I specific carbohydrate but failed to ef- 
fect a complete decomposition of that substance, since it never 
fully lost its precipitating ability with homologous serum. Sickles 
and Shaw suggested that this residual reaction was due to an un- 
used portion of the original carbohydrate or to products of de- 
composition, which might either be present in the original sample 
or be formed as a result of the action of the microorganism from 
the soil. Another possibility presents itself and that is that the au- 
thors' carbohydrate preparation may have contained a fraction 
which was not susceptible to the enzymatic action of the culture. 
It was impossible to obtain any soluble enzyme from this organism. 

Several of Sickles and Shaw's cultures decomposed the non- 
type-specific carbohydrate isolated by Wadsworth and Brown (the 



POLYSACCHARIDE-SPLITTING ENZYMES 319 

C Fraction) from an attenuated strain of Type I Pneumococcus. 
One organism was the aerobic, spore-forming bacillus that had 
been found to utilize the soluble specific substance of Type III 
Pneumococcus and agar; the other was a Gram-negative aerobic, 
non-motile, non-sporulating bacillus which, so far as tested, di- 
gested only this non-type-specific saccharide. From both of the 
strains a soluble enzyme was obtained capable, like the living cells, 
of splitting the non-type-specific carbohydrate and, in doing so, of 
abolishing its power to produce purpura. 

Sickles and Shaw 1282 then undertook a systematic study of the 
morphological, cultural, and biochemical characters of these vari- 
ous polysaccharide-decomposing bacteria. The cultures had origi- 
nated in the muck of swamps and in uncultivated soils of different 
localities, while one strain came from manure. The organisms were 
grown on the mineral medium of Dubos and Avery and a "Medium 
S" of their own concoction. To these mineral media were added the 
specific pneumococcal carbohydrates in concentrations varying 
from 0.002 to 0.01 per cent as a source of carbon. 

Sickles and Shaw then subjected the strains to all the proce- 
dures required for identification, and in their communication gave 
a detailed account of the different biological characters of the or- 
ganisms. To relate them would not be germane to the main subject 
but, because of the importance of this class of bacteria, a con- 
densed description of the main characters should be recorded here. 
The cultures were of four distinct types: 

1. Large spore-bearing rods that decompose the specific carbohydrate 
of Type III Pneumococcus, similar to the SIII bacillus of Dubos and 
Avery, and, in addition, a strain that utilizes agar. For the strains 
that digest only the Type III polysaccharide the authors suggested 
the name Bacillus palustris, with the sub-designation, gelacticus, for 
the variety also attacking agar. 

2. Very small non-sporulating rods that attack the non-type-specific 
carbohydrate obtained from a degraded Type I Pneumococcus and the 
C Fraction from typical strains. This organism the authors called, 
Flavo-bacterium ferruginum. 



320 BIOLOGY OF PNEUMOCOCCUS 

3. Bacteria, oval in form, that decompose the specific carbohydrate 
of Type II Pneumococcus. To these organisms was given the species 
name of ovale and the designation Saccharobacterium was proposed for 
this new genus of the family Mycobacteriaceae including the microor- 
ganisms decomposing the specific carbohydrates of Pneumococcus of 
Types I and II. 

4. Slender rods with tapering ends that utilize the specific carbohy- 
drate of Type I Pneumococcus, for which the authors suggested the 
name Saccharobacterium acuminatum. 

Again from soil, Sickles and Shaw 1284 isolated another strain of 
Bacillus palustris, which through the agency of a soluble enzyme 
decomposed the specific carbohydrate of Type VIII Pneumococ- 
cus. The enzyme was apparently without action on organisms of 
Types I, II, or III. Although Type VIII specific carbohydrate 
gave marked precipitation with Type III antiserum and, con- 
versely, Type III carbohydrate precipitated Type VIII antise- 
rum, it was a curious fact that the culture or enzyme that decom- 
posed Type III carbohydrate failed to attack the polysaccharide 
of Type VIII, and the enzyme decomposing Type VIII carbohy- 
drate was without action on the carbohydrate of Type III pneu- 
mococci. Here the enzymatic selectivity appears to be even greater 
than the overlapping serological specificity exhibited by chemically 
related but not structurally identical carbohydrates. 

DIFFERENCES IN SUSCEPTIBILITY OF POLYSACCHARIDES TO ENZY- 
MATIC ACTION 

A possible explanation for the differences in the susceptibility of 
pneumococcal polysaccharides to enzymatic action and in their 
serological behavior is to be found in the recent study of Goebel 
(1935). 519 Quoting from his communication: 

In the present study the specific polysaccharide of Type VIII pneu- 
mococcus has likewise been shown to be constituted from molecules of 
glucose and glucuronic acid. After hydrolysis with dilute mineral acid 
there appear in the hydrolysate of the carbohydrate from Type VIII 



POLYSACCHARIDE-SPLITTING ENZYMES 321 

pneumococcus a hexose, identified as glucose, and an aldobionic acid. 
From the actual quantities of these two constituents found in the hy- 
drolysate, and from the value of the acid equivalent of the polysaccha- 
ride itself, it appears that this specific carbohydrate is built up from 
glucose and glucuronic acid approximately in the ratio of 7 molecules of 
hexose to 2 of uronic acid. The capsular carbohydrate of Type VIII 
pneumococcus represents, therefore, an entity which is chemically dis- 
tinct from the specific polysaccharide elaborated by the Type III pneu- 
mococcus. 

That a chemical similarity between the two substances exists, how- 
ever, may be seen from the results of the experimental work in which it 
has been shown that the aldobionic acids appearing in the acid hydroly- 
sates of both polysaccharides are identical. The proof of the identity of 
these two uronic acids was made possible through the preparation of the 
crystalline heptaacetyl methyl ester. Both derivatives show identical 
crystalline structure, melting points, and specific rotations. Further- 
more, a mixed melting point of the two derivatives shows no depres- 
sion. Although the actual structure of the aldobionic acid is as yet un- 
known, work is now in progress to establish this point. 

In view of the experimental evidence which has been presented, 
showing that the aldobionic acids derived from the carbohydrates of 
Types III and VIII pneumococcus are identical, it is believed that the 
basis for the immunological crossing exhibited by these two specific 
types of pneumococcus resides in the structural and configurational 
identity of the uronic acid nucleus common to the encapsulating poly- 
saccharides of both microorganisms. 

Sickles and Shaw 1283 reported that the enzyme from the new soil 
bacillus was capable of destroying the purpura-producing action 
of the VIII carbohydrate and of protecting mice against several 
thousand minimal fatal doses of a virulent strain of Type VIII 
Pneumococcus. Larger amounts of the enzyme failed to protect 
mice against ten minimal fatal doses of Type III Pneumococcus. 
The observations in these two papers describing the highly selec- 
tive action of the enzyme on the soluble specific substance in con- 
trast to the cross-agglutination present another pretty problem 
for the chemo-immunologist. 



322 BIOLOGY OF PNEUMOCOCCUS 

SUMMARY 

The discovery of bacteria endowed with enzymes capable of 
breaking down the complex polysaccharides of Pneumococcus is of 
more than academic interest. Here is a biological agent that in no 
way interferes with the vital activities of the pneumococcal cell; 
the cell grows, multiplies, and continues to synthesize the complex 
capsular carbohydrate peculiar to its serological type, but as soon 
as the carbohydrate is formed it is stripped from the coccus by the 
digestive action of the alien bacterial enzyme. The ability of a mere 
saprophyte to rob a pathogen of its protective covering, and 
thereby to deprive it of its infective power, is one of the strangely 
fascinating surprises of bacteriological research, but it has a much' 
deeper meaning. The nature of this enzymatic action is in accord 
with the specific chemical constitution and serological behavior of 
the specific capsular polysaccharide of the pneumococcal type af- 
fected. By denuding the cell of its capsule through the dissolution 
of its polysaccharide, the enzyme exposes the cell to the destruc- 
tive action of the white corpuscles of the blood, and if the tissue 
cells are functionally vigorous enough, the attack of the invading 
pneumococci is repelled and the infection aborted. 

The enzymes of certain of these bacterial forms have been shown 
to save mice, rabbits, and monkeys from all but a massive infection 
from Pneumococcus and, what is more striking, the enzyme of one 
bacterial species directs its attack against the members of Type 
III, for which, as yet, no biological curative agent has been de- 
vised. The possibilities presented by these recent disclosures en- 
gage the imagination. The bacteriologist will want to search for 
still other microbic cells with enzymes capable of consuming the 
carbohydrates of pneumococci of the other serological types ; the 
biochemist will desire to know more of the nature of the enzymatic 
principle existing in these bacterial cells ; while the investigator in 
immunology will follow this inviting lead toward a resolution of 
some of the processes operating in infection and resistance. 



CHAPTER X 
ANTIGENICITY OF PNEUMOCOCCUS 

The immunizing properties of Pneumococcus and of its compo- 
nents and derivatives; factors which operate in establishing the im- 
mune state; and the response of different animals to the antigenic 
action of members of this bacterial species and their constituents. 

IN Chapter VIII the antigenic properties of the chemical con- 
stituents of Pneumococcus were described only in a general way 
in order to illustrate the similarities and dissimilarities of the pro- 
tein and carbohydrate preparations which have been advanced as 
representing the active substances native to the cell, and to empha- 
size the dependence of immunological behavior on chemical consti- 
tution. No attempt was made to give any systematic account of 
the various antibodies or immune effects which the proteins and 
polysaccharides call forth, or to compare and correlate the action 
of the cellular fractions with that of the intact bacterial body. 

Antigenic Spectrum 

The antigenic spectrum of Pneumococcus can be resolved into 
its dominant bands in several ways. Taking the whole, living, fully 
virulent cell as the norm, its immunological action can be analyzed 
through the stimuli provided by its separate constituents or de- 
rivatives. There enter into the analysis the chemical structure of 
the several components of the cell, the question of their mass, and 
the route and spacing of their administration. 

The spectral bands — or less definitely, zones or regions — ap- 
pearing after the introduction of pneumococcal materials into the 
animal body, are represented by agglutinins, precipitins, bacteri- 
cides, tropins or opsonins — conceivably the alleged antitoxins — 
the complement-fixing and protective antibodies, and such manifes- 



324 BIOLOGY OF PNEUMOCOCCUS 

tations of somatic reactivity as systemic or local allergy. All the 
phenomena depend in turn on the inherent special or racial pe- 
culiarities of the animal subject. 

The resolution of the antigenic spectrum involves the study of 
the effects of Pneumococcus itself, of its separate constituents with 
their individual, chemical identification, in their relation to the 
parent material; the diverse serological reactions in which the 
whole cell or its parts participate ; and the nature of the mechan- 
ism of the particular physiological response each wakens. 

These ways bristle with obstacles of one kind or another. There 
is the tendency of Pneumococcus to digest itself or to lose its 
vigor; there is the danger of fracturing the molecular mosaic of 
the cellular substances by chemical treatment; there are all the 
subtle factors which influence the interaction of antigen and anti- 
body in the test tube ; and then there is the constitutional capacity 
or incapacity of animal tissues to function in response to the intro- 
duction of alien substances into the body. An antigen by one test 
may display full power ; by another test it may seem to be incom- 
plete or inert. Introduced in inappropriate quantity, an antigen 
may defeat its effect ; administered by one route we see one set of 
immune bodies, by a different route some of the antibodies may ap- 
pear wanting type-specificity or else be entirely absent; injected 
into animals of one species an antigen provides the animal with a 
full complement of antagonistic substances and protects it against 
fatal infection, while in other animals the administration of the 
same antigenic substance may be followed by no appreciable im- 
mune effect. Here, not only the race but the lineage, the age, and 
the immediate physical state of the animal all have their part in 
deciding the issue. Antigens and antigenicity, like virulence, there- 
fore, are purely relative terms. 

First Observations of Immunity 

For a complete and orderly presentation of the manifestations 
of the immunological behavior of Pneumococcus it seems best to 



ANTIGENICITY OF PNEUMOCOCCUS 325 

develop the subject by showing the manner in which our present 
knowledge has been acquired. Although it was Fraenkel 468 who first 
observed that rabbits surviving a subcutaneous injection of pneu- 
mococci became resistant to subsequent inoculation with the same 
organism, it was Foa and Bordoni-Uffreduzzi 461 who established 
the fact in an experimental way. By injecting rabbits subcutane- 
ously at three or four-day intervals, beginning with attenuated 
cultures and then giving cultures of increasing virulence, the ani- 
mals became insusceptible to inoculation with virulent cultures or 
infected blood. The authors applied the method to man but with 
no success. Then Foa 458 turned to the soluble products of Pneumo- 
coccus, and attempted the chemical isolation of the immunizing 
principle from broth cultures, but the experiments were inconclu- 
sive. 

A great debt is owing the Klemperers, 723 ' 5 who laid the founda- 
tion for the active immunization of animals against pneumococcal 
infection. As has already been told, the Klemperers used the spu- 
tum of convalescent pneumonia patients, bacteria-free pleural 
exudates, heated glycerol extracts, and heat-killed cultures as an- 
tigens and hence became the first to demonstrate that dead pneu- 
mococci and some of their derivatives, quite as well as the living 
cells, can render an animal immune. They also introduced the in- 
travenous route for the administration of antigenic materials and 
proved that the serum of the immunized rabbits carried substances 
antagonistic to Pneumococcus. Like Foa, the Klemperers believed 
incorrectly that the immunizing principle in Pneumococcus was a 
toxin. 

There then began a succession of publications dealing with the 
action of various pneumococcal materials in inducing immunity. 
There were the successful results of Emmerich and Fowitzky 357 
with cultures of attenuated and then of diluted, virulent strains, 
and those of Bonome, 137 and of Kruse and Pansini, 763 with sterile 
culture filtrates, injected intravenously, subcutaneously, and in- 
traperitoneally. Mosny 932 was another to employ sterile culture fil- 



326 BIOLOGY OF PNEUMOCOCCUS 

trates, while Foa and Carbone 463 obtained a certain degree of 
immunity with alcohol and ammonium sulfate precipitates from 
culture filtrates. The antigenic action of these agents was feeble, 
but Foa and Carbone demonstrated that both the intact cell, living 
or dead, and substances elaborated by the cell during cultivation 
evoke an immune response. The work of Denys 312 and of Wash- 
bourn 1486 established the fact that in order to produce immunity of 
high grade it was essential that pneumococci, whether used in the 
living or dead state or in the form of heated or filtered cultures, 
should be virulent. 

These were the beginnings of immunizing procedures which have 
become routine, but there are many features of the subject that 
deserve more detailed discussion. The use of pneumococci taken 
from growths on solid media or in broth for the production of im- 
mune serum for experimental purposes has long been the common 
custom. For some time it was believed that for the production of a 
potent serum it was necessary to inject living organisms, but ex- 
perience has shown that response to antigenic stimulus, that is the 
production of a serum with a high content of strictly type-specific 
antibodies, is conditioned less by the viable state of the organism 
than by the biological state of the culture at the time it is em- 
ployed as antigen. 

Influence of Virulence on Immunological Response 

It is now generally accepted that for the production of a high 
degree of type-specific immunity the antigen, whether in a living or 
devitalized condition, must come from a robust, virulent culture. 
This conviction arises from the results of experiments on animals 
with strains possessing varying degrees of invasiveness, from the 
fully virulent, smooth forms to the degraded, rough forms, and 
from the experience of those workers who are engaged in the manu- 
facture of therapeutic serum. The reason supporting the convic- 
tion is the chemical demonstration that complete antigenicity is 



ANTIGENICITY OF PNEUMOCOCCUS 327 

dependent upon the maximal amount of unaltered protein and car- 
bohydrate in the cell. 

All through the literature on pneumococcal immunity, from the 
time of Denys and of Washbourn, there are repeated statements to 
the effect that virulent pneumococci are more active immunizing 
agents than are attenuated strains. The study of the antigenic ac- 
tion, especially in regard to type-specificity, of variant forms of 
pneumococci substantiates this fact. Neufeld, Cole, Wadsworth, 
and others, have always advocated the use of cultures in a virulent 
state for the production of immune serums. Barach 75 has shown 
that a highly virulent pneumococcus provokes a more marked im- 
munity than an organism of low virulence — an observation cor- 
roborated by Meyer and Sukneff, 898 Day, 308 and many others. 
Therefore, whether the immunizing antigen is to be the living or 
killed organism or any of the separate substances isolated or de- 
rived from the cell, the evidence is wholly in favor of selecting a 
culture in vigorous condition and of exalted virulence. 

Dead Cultures 

HEAT-KILLED ANTIGENS 

For a long time living pneumococci, usually after a preliminary 
course of injections of heat-killed cultures, were used for the vac- 
cination of horses in the production of curative serums. The prac- 
tice, however, caused a grave mortality among the animals under 
treatment. In 1917, the senior author of this volume, among 
others, desiring to prevent the depletion of his stables, substituted 
for living cocci, heat-killed organisms from broth cultures, and al- 
though the agglutinin titer of the horses was low, the content of 
the serum in mouse-protective antibodies was more than sufficient 
to meet standard requirements of that time. 

Using cultures devitalized by heat, formaldehyde, soaps, or 
other means became the practice in many serum-producing labora- 
tories, although the literature contains relatively few detailed ob- 



328 BIOLOGY OF PNEUMOCOCCUS 

servations on the comparative antigenic action of pneumococci 
when in the living and in the dead state.* In 1902, Neufeld 974 ob- 
tained satisfactory agglutinating serum by the injection of rabbits 
with either living or heat-killed pneumococci, but for the routine 
preparation of immune serum he gave the animals, first, heat-killed 
cultures, and then living cultures. Wadsworth, 1457 in 1912, claimed 
that only after immdnization with living, virulent cultures did the 
serum acquire marked curative properties. 

Cotoni and Brasie 281 made a comparative study of the immuniz- 
ing effect of pneumococci heated at 56°, of cultures treated with 
alcohol and ether and suspended in salt solution, and of these sus- 
pensions heated for fifteen minutes at 110°. The heat-killed cocci 
proved to be far superior to the other antigens in protecting rab- 
bits against infection. 

The influence on antigenic action of varying exposures of pneu- 
mococci to different temperatures was investigated by Tani. 1379 Ac- 
cording to the results obtained, cultures of Type I pneumococci 
incubated for a long period at 39°, when injected intraperitone- 
al^ into mice, were found to have lost virulence and were feeble 
immunizing agents ; cocci heated for two and one-half hours and 
not completely killed showed poor immunizing power, whereas an- 
other specimen heated at the same temperature for an equal length 
of time and completely killed, and then later heated for one-half 
hour at 56°, gave good protection. After subjecting a similar cul- 
ture to a four hours' exposure at 100°, it was still antigenic in 
that it afforded protection to mice. Tani's results point to viru- 
lence as an essential property of cultures used for immunization. 
Davidson, 298 too, tested the antigenic action of living pneumococci, 
of heat-killed cultures, and of detoxified and defatted vaccines. 
Rabbits injected with the killed cultures acquired immunity, and 
the serum contained protective substances. The living cultures pro- 

* These facts and many similar observations by others have never been pub- 
lished but are widely current in the stable lore of manufacturing laboratories. 



ANTIGENICITY OF PNEUMOCOCCUS 329 

duced intoxication, while the use of acetone and other agents in 
removing the lipids from the vaccine destroyed its antigenic power. 

In the case of Type III Pneumococcus, Tillett 1400 found that re- 
peated injections of heat-killed cultures into rabbits were effective 
in producing active immunity against infection with a virulent 
strain of the homologous type. Even heterologous strains, de- 
vitalized by heat, were capable of affording similar protection to 
Type III cocci. Moreover, this form of active immunity could exist 
in the absence of demonstrable type-specific antibodies and appar- 
ently was unrelated to the variety of Pneumococcus used for im- 
munization. Tillett considered that this anomalous immune state 
was dependent upon the exaltation of the same factors which af- 
ford normal rabbits material resistance to some strains of Type 
III organisms. 

Employing Type II strains, Gaspari, Sugg, Fleming, and 
Neill 503 learnt that the continued injection of heat-killed cultures 
of this type led to a decrease in type-specificity and to an increase 
in the species-specificity of the serum of the treated rabbits. For 
the preparation of diagnostic serum of high type-specificity the 
authors preferred as an antigen the heated cells of virulent pneu- 
mococci administered over a comparatively short immunization pe- 
riod. A gradual shift from type-specificity to species-specificity is 
no uncommon occurrence in horses that have been under immuni- 
zation for long periods of time with either living or dead organisms. 

Day 307 reported that it was generally desirable to kill the cells 
with heat for the production of antibodies, but warned against the 
use of a temperature as high as 100° for the purpose, since such a 
degree of heat destroyed the antigenic properties of the culture. 
However, this has not been the experience of the authors of the 
present volume. As might be expected, Day, and also Harley, 592 
found that any agent which promoted the digestion of the cell, and 
which consequently led to the breakdown of its constituents, weak- 
ened or finally abolished the ability to call forth type-specific anti- 



330 BIOLOGY OF PNEUMOCOCCUS 

bodies. Additional evidence confirming the efficacy of heat-killed 
antigens is to be found in the experiments of Stillman and Good- 
ner. 1342 Three intravenous injections at four-day intervals of saline 
suspensions of heat-killed cultures of Type I, II, and III pneumo- 
cocci protected rabbits against an otherwise fatal infection of 
pneumococci of the corresponding type administered according to 
Goodner's intradermal technique. The results were controlled by 
the demonstration of agglutinins and protective antibodies in the 
serum of the treated animals. 

In 1923, Yoshioka, 1562 finding that the successful immunization 
of mice was largely dependent upon the total mass as well as the 
spacing of the doses of antigen, devised a rapid method involving 
small quantities of heat-killed pneumococci injected in six half- 
hourly doses. In this way he obtained excellent protection in mice. 
In the next year, Killian 704 repeated Yoshioka's experiments with 
intensive serial injections and, in addition, tried injections of 
killed cultures with weekly intervals between each series. By this 
plan of three series of intraperitoneal injections at weekly inter- 
vals, it was possible to render mice immune to a fatal dose of pneu- 
mococci given three days after the completion of the immunizing 
treatment, but the immunity lasted only a few weeks. Killian laid 
stress on the proper amount of antigen to be given, because doses 
too small or too large yielded poorer results. The use of living, 
fully virulent organisms, contrary to the experience of most ob- 
servers, resulted in a noticeably lower degree of immunity. 

In another communication, Killian 706 gave the details of further 
experiments from which it appeared that by employing the intra- 
venous route for serial injections the level of protection in mice 
could be elevated, and that by periodic intraperitoneal treatment 
of the animals with four-day intervals between injections of in- 
creasing doses of killed vaccine one could obtain a high degree of 
protection in a short time. In a third communication, Killian 707 de- 
scribed a refractory stage which lasted for several days after an 
immunizing injection and, in order to avoid this stage and to se- 



ANTIGENICITY OF PNEUMOCOCCUS 331 

cure the most effective secondary stimulus, gave injections at in- 
tervals of fourteen instead of four or eight days. 

The question of the effect of heat on the immunizing activity of 
Pneumococcus was subjected to systematic study by Barnes and 
White, 86 who compared the antibody response following the injec- 
tion into rabbits of heat-killed and formalinized cultures of Type I 
Pneumococcus, administered according to different schedules over 
long periods — in some of the experiments for as long as eight 
months. As antigens, the authors used saline suspensions of the 
sediment from eighteen-hour broth cultures of a virulent Type I 
strain, subjected to 56° in a water-bath for one hour, and similar 
suspensions of culture sediment treated with 0.3 per cent formalin. 
The vaccines were standardized by the Gates 504 nephelometer and 
the individual doses accurately measured. The injections were given 
intravenously according to three different plans somewhat similar 
to those of Yoshioka and of Killian. The schedules consisted re- 
spectively of a single injection on each of three successive days; a 
single injection on each of five successive days ; and a series of six 
injections on each of the first two days with the other four on the 
third day. The rest periods varied from one to three weeks, and 
test bleedings were usually taken seven, twelve, and, in some in- 
stances, twenty-two days after the last injection of a series. The 
various serum samples were titrated for agglutinins, precipitins, 
and mouse-protective antibodies. 

Heat-killed pneumococci produced highly potent serums of strict 
type-specificity. The first and third named methods were equally 
productive, but the first was the method of choice because of its 
greater simplicity. The results of the study, besides confirming the 
value of heat-killed pneumococci in establishing a high degree of 
immunity, afforded a rational basis for the immunizing treatment 
of horses for the production of therapeutic serum. 

OTHER DEVITALIZED ANTIGENS 

As soon as it was found that heat-killed pneumococci were capa- 



332 BIOLOGY OF PNEUMOCOCCUS 

ble of calling forth a specific immune response, other attempts 
were made to rob the cell of the power of invasion and infection 
without impairing its antigenic qualities. For a long time for- 
maldehyde in the form of formalin has been employed for the pur- 
pose of killing and preserving pathogenic organisms, but it is not 
clear when it was first used to prepare pneumococci for the pur- 
pose of immunization. Tao (1932) 13so reported that the antigenic 
potency of formalinized cultures was greater than that of heat- 
killed cultures in causing the development of agglutinins and pro- 
tective antibodies in rabbits and mice. Formalin in a concentration 
of 0.3 per cent was added to the cultures and the treatment of the 
animals was limited to one subcutaneous and two or three intra- 
venous injections. 

Pico and Negrete 1089 prepared immunizing antigens for horses 
with 0.05 per cent formalin. The authors attributed the keeping 
qualities of the vaccine to the action of formaldehyde in rendering 
the soluble specific substance insoluble in water, but it has been the 
experience of the writers of the present volume that concentrations 
of formalin as high as 0.3 per cent fail to prevent the gradual lysis 
of pneumococci suspended in salt solution. In Ferguson's 436 experi- 
ence in actively immunizing white mice and rabbits with both heat- 
killed and formalinized vaccines, it appeared that the latter were 
only slightly superior to heat-killed cultures. Formalin was used in 
a strength of 0.2 per cent, and suspensions were made in salt solu- 
tion containing 0.5 per cent phenol. There was a tendency of killed 
pneumococci to autoWze with a loss of antigenic power accom- 
panying the disintegration of the cell. For this reason, as well as 
because of their better immunizing effect, Barnes and White gave 
their choice to heat-killed vaccines, since these vaccines undergo 
less deterioration than those prepared with formalin. 

According to Dubos,* formalin added in a concentration of 0.3 
per cent to suspensions of living pneumococci prevents multiplica- 
tion but fails to inhibit the activity of intracellular enzymes. The 

* Personal communication, 1936. 



ANTIGENICITY OF PNEUMOCOCCUS 333 

concentration of formalin must be raised to 0.5 per cent if enzy- 
matic activity is to be destro} r ed. On the other hand, exposure of 
the pneumococcal suspensions to heat, when properly carried out, 
yields a vaccine of great stability and of high antigenic integrity. 
Dubos observed that exposure to temperatures that rob the cell of 
its ability to grow and multiply may fail to halt the action of the 
protease of Pneumococcus. To devitalize the organisms, to prevent 
autolysis, and to preserve the antigenic properties of pneumococci, 
Dubos heated cultures or suspensions to 75° with the least pos- 
sible delay and so obtained antigens of greater stability and im- 
munizing power than could be produced by any other method. 

Confirming the claims of Barnes and White and of Dubos that 
immediate heating of pneumococci for use as antigens is the best 
method for insuring the stability of the preparation, O'Meara and 
Brown, 1031 during the course of an investigation on the readiness 
with which pneumococci disintegrate, reported that, in spite of a 
large number of preservatives tried, no agent except heat was 
found that would completely arrest autolysis of pneumococcal cul- 
tures. 

Day 307 used formol, or antiformin, as a killing agent, but aban- 
doned its use since it weakened the immunizing properties of the 
cultures. Truche 141920 favored an alcohol-ether mixture for the 
routine preparation of antigens in the production of equine anti- 
pneumococcic serum, and claimed that cultures so treated, dried 
in a vacuum, and suspended in salt solution, were tolerated better 
by horses than were heat-killed cultures. 

There have been reported many other methods for rendering 
pneumococci harmless for immunizing purposes. As early as 1902, 
Sergent 1206 treated suspensions of agar cultures of pneumococci 
with crystal violet. Although the dye in the concentration used 
failed to kill the cocci it effected a partial attenuation, so that rab- 
bits surviving the intravenous or intraperitoneal injection of the 
treated cultures were able six to eight days later to resist increased 
doses of the same cultures that proved fatal in animals which had 



334 BIOLOGY OF PNEUMOCOCCUS 

not received a previous injection of the dyed cocci. Sergent's appli- 
cation of coal-tar dyes to pneumococci for the purpose of prepar- 
ing immunizing antigens appears to be a lone observation of its 
kind. 

Nicolle and Adil-Bey (1907) 1007 obtained immunity in rabbits 
by the injection of cultures treated with sodium choleate, while 
Meyer (1927) 897 and later Meyer and Sukneff (1928) 898 reported 
that Type I pneumococci dissolved in sodium taurocholate would 
immunize both rabbits and mice. The serum of the rabbits so 
treated contained a moderate amount of protective substance but, 
as might be expected, the serum as a rule gave some cross-protec- 
tion against Type II strains. While pneumococci treated with bile- 
salts may induce immunity in mice and rabbits and presumably in 
other animals, the immunity is of a low order and is lacking in 
type-specificity. As late as 1933, Ziegler 1573 advocated as an immu- 
nizing agent "Pneumocholin," a substance of unknown chemical 
composition produced by the lysis of pneumococci in sodium 
taurocholate solution. The substance was stable under refrigera- 
tion and, when injected into rabbits, induced after three or four 
days an effective immunity to Type I infection. 

Larson and Nelson 791 recommended the use of sodium ricinoleate 
in the preparation of pneumococcal vaccines both for prophylactic 
use in man and for the preparation of therapeutic serum. A viru- 
lent culture of Pneumococcus treated with ricinoleate soap in a 
final concentration of 0.1 per cent immediately lost its patho- 
genicity and, according to the authors, when injected intraperi- 
toneally into rabbits, stimulated the production of large amounts 
of agglutinins within twenty-four hours. The animals resisted 
many multiples of the infective dose of pneumococci, and their se- 
rum protected normal rabbits against both intravenous and intra- 
peritoneal infection. The serum of rabbits and sheep immunized 
with the sodium ricinoleate antigens was claimed to possess dis- 
tinct curative properties for rabbits infected with Pneumococcus. 
The administration of the immune serum to patients suffering 



ANTIGENICITY OF PNEUMOCOCCUS 335 

from lobar pneumonia was followed by a rapid drop in tempera- 
ture and relief of subjective symptoms. Larson believed that the 
effect was due to the presence of antitoxin in the serum. 

In a later communication, Larson 789 reported that the intraperi- 
toneal injection of large amounts of soaped pneumococci of Types 
I, II, and III protected rabbits against infection induced by the 
Goodner technique, and claimed that by this method it was possi- 
ble to produce both species-specific and type-specific immunity. 

Sensitized Pneumococci 

The use of pneumococci sensitized with homologous immune se- 
rum was described by Levy and Aoki (1910), 801 while Alexander, 7 
after similarly treating and incubating the cocci with leucocytes, 
could immunize rabbits with the preparations. Protective sub- 
stances appeared in the serum of the animals within eight to eleven 
days after the first injection, but the method possessed no advan- 
tages over the use of heat or formalin for the preparation of anti- 
gens. Levy and Aoki, and also Killian, 704 employed phenol as a 
devitalizing agent, but the latter author found that phenolized 
vaccines showed a definite loss of immunizing power after six weeks' 
preservation. 

Filtrates and Extracts 

CULTURE FILTRATES AND BACTERIAL EXTRACTS 

Early investigators turned to culture filtrates and to other 
products and derivatives of Pneumococcus in a search for some 
principle which would increase the resistance of experimental ani- 
mals and eventually of man to pneumococcal infection. 

Warden (1912) 1485 claimed to have immunized rabbits with pan- 
creatic extracts of pneumococci and made the additional observa- 
tion, which apparently has never been confirmed, that similar ex- 
tracts of staphylococci and pancreatic extracts alone were capable 
of inducing immunity in infected and non-infected rabbits, and of 



336 BIOLOGY OF PNEUMOCOCCUS 

favorably modifying the course of pneumococcal infections in man. 
However, in Warden's communication no experimental data were 
presented to support these unusual claims. Aitoff and Lagrange 
(1925) 6 stated that an injection of sterile medium T made at 
least twenty-four hours previous to inoculation with Pneumococ- 
cus conferred protection upon mice which was demonstrable for at 
least two weeks after injection of the medium. 

In 1926, Horder and Ferry, 655 in a search for an ideal antigen 
for therapeutic immunization, measured the agglutinin content 
and complement-fixing titer of the serum of rabbits treated respec- 
tively with washings from agar cultures, autolysates of the residue 
from broth cultures, washed agar growth, and whole unwashed 
agar cultures. Horder and Ferry concluded that the material con- 
tained in the washings of agar cultures, because it could be pre- 
pared in watery solution and was low in protein content, as well as 
being practically non-toxic and high in antigenic properties, more 
nearly approached the ideal antigen than any preparation which 
had come under their observation. Barach (1928) 74 " 5 reported that 
Berkefeld filtrates of pneumococci shaken in salt solution and also 
filtrates of broth cultures caused in mice the development of pro- 
tection which appeared on the fourth day after injection. The im- 
munity produced was dependent upon type-specific protective sub- 
stances and not upon the elaboration of the common protein anti- 
body. 

Another method of preparing antigens from broth cultures of 
pneumococci was that described by Maeji (1930), 850 ' 853 ~ 4 who em- 
ployed the technique of Besredka for preparing antivirus. The su- 
pernatant fluid from centrifuged beef-serum bouillon cultures of 
Type I Pneumococcus, when injected subcutaneously into rabbits, 
gave partial protection against infection with a Type I strain. In 
the case of Type II Pneumococcus the protective substance ap- 
peared only when goat serum was added to the medium, while with 
Type III organisms no antivirus could be obtained. Maeji claimed 
that after the administration of Type I antivirus by inhalation, 



ANTIGENICITY OF PNEUMOCOCCUS 337 

young rabbits resisted intratracheal inoculation with a virulent 
culture, and that resistance could be increased by repeated in- 
halation of the preparation. The attenuating action of filtered 
broth cultures of pneumococci on fresh cultures of the same or- 
ganism was reported by Okischio (1932), 1023 but the use of anti- 
virus either for the preparation of vaccines or for the treatment 
of pneumococcal infections has not come into general use. 

There are yet other references to the use of extracts and au- 
tolysates of Pneumococcus for the purpose of inducing immu- 
nity. Among them might be mentioned those of Day (1930 and 
1934 ) 307 ' 309 who reported that the intraperitoneal injection into 
mice of autolysates of both Type I and Type II cultures was fol- 
lowed one week later by protection against one thousand to ten 
thousand fatal doses of culture. Another communication was that 
of Harley (1934) 591 in which was reported the preparation of an- 
tigens by extracting Type I and II pneumococci with 0.05N hy- 
drochloric acid at 60°. 

TISSUE EXTRACTS AND EXUDATES 

The possibility that products arising from the vital activities of 
Pneumococcus in animal tissues might exert immunizing action and 
that such products might be applied to the protection or treat- 
ment of animals and human beings has not been neglected. Foa 
and Carbone, 463 Bonome (1891), 137 and Mosny (1892) 932 were 
thus able to render rabbits relatively invulnerable to the injection 
of otherwise lethal doses of pneumococcal cultures. Hartman 
(1913) 598 studied the antigenic action of the principal constituents 
of pneumonic exudates, but the results were largely negative. 
Freedlander (1928) 480 employed saline extracts of organs of in- 
fected rabbits as antigens and, after three injections at five to 
seven-day intervals into normal rabbits, obtained a serum of high 
protective titer for mice against Type I Pneumococcus and a se- 
rum of somewhat less potency against a Type II culture. While 
the serums compared favorably in protective action with thera- 



338 BIOLOGY OF PNEUMOCOCCUS 

peutic Type I antipneumococcic serum, they were low in agglu- 
tinins, contained no precipitins, possessed no remarkable opsonic 
powers, and displayed no direct bactericidal action. 

The idea was revived in 1930 by Banzhaf and Curphey, 71 who 
combined in horses intramuscular injections of phenolized pneu- 
mococcal pleural exudates with intravenous injections of the for- 
malinized sediment from broth cultures of pneumococci. When 
tested by Goodner's method the therapeutic value of the serum was 
disproportionate to the mouse-protective action, which fact Banz- 
haf and Curphey interpreted as indicating the possible presence 
of added therapeutic substances in the serum resulting from the 
addition of exudate to the usual method of immunization. 

Toxins and Hemotoxins 

SO-CALLED TOXINS 

The possible existence of toxins either in the pneumococcal cell 
or resulting from its metabolic processes has already been dis- 
cussed in Chapter III. It is extremely doubtful if Pneumococcus 
possesses or produces a true toxin, but the idea still survives, and 
the action of some therapeutic serums, quite apart from their 
tropic, agglutinative, precipitative, or protective effects, in amel- 
iorating the intoxication of pneumonia in man, has encouraged 
the search for a toxic principle with the hope of producing an 
antitoxin. 

The Klemperers (1892) 725 were convinced that they had suc- 
ceeded in isolating a toxic protein from Pneumococcus, which they 
named "Pneumotoxin," but the action of the substance can now be 
explained on other grounds. In 1897, Auld 30 separated an albu- 
mose and an organic acid from local lesions, lungs, and spleens of 
animals infected with Pneumococcus. No adequate proof could be 
obtained that the protein was a true toxin, but Auld intimated that 
a true toxin might in all probability be united with the protein. 
The albumose preparations had a certain immunizing value, but no 



ANTIGENICITY OF PNEUMOCOCCUS 339 

evidence was advanced to show that the immunity was antitoxic in 
nature. 

In 1912, Rosenow 1168 obtained from pneumococci a poisonous 
substance, similar to histamine in its action, but wholly incapable 
of provoking any immune response. Larson, 788 and then Olson 
(1926), 1029 believed that Pneumococcus contained a toxin and that 
by injecting animals with whole cultures of the organism treated 
with sodium ricinoleate, or with autolyzed broth cultures, it was 
possible to produce an antitoxic serum. The assumption was based 
solely on the physiological effect of the serum and not on any 
demonstration or titration of the antitoxin which the serum was 
supposed to contain. 

The work of Parker 1061 and of Parker and McCoy (1929) 1062 is 
more suggestive. By allowing pneumococci of Types I, II, and III 
to autolyze under strict, anaerobic conditions, the authors ob- 
tained a preparation that was toxic for guinea pigs and for horses 
and which, when injected repeatedly into horses over a period of 
eight months, caused the animals so treated to develop in the serum 
substances capable of neutralizing in vitro the toxic autolysates. 
Parker reported that antipneumotoxic serum produced in rabbits 
or horses by immunization with sterile filtrates of pneumotoxin af- 
forded heterologous protection to guinea pigs — at least as far as 
Type I or Type II pneumococci were concerned — against the de- 
velopment of pneumonia, while the serum of some of the treated 
horses exerted a curative action against infection with pneumo- 
cocci of the homologous type, but contained little if any antitoxin 
for heterologous types. Furthermore, since the serums contained 
no specific protective antibodies against pneumococci, Parker con- 
cluded that the serum was antitoxic in action. 

The experiments of Parker and McCoy, including a definite de- 
termination of the toxic power of sterile pneumococcal autolysates 
and of the neutralizing strength of serum produced in response to 
the injection of the autolysates, with evidence of the absence of 
specific protective antibodies, constitute the first accurately con- 



340 BIOLOGY OF PNEUMOCOCCUS 

trolled observations of the kind and are sufficiently suggestive to 
encourage further study of pneumococcal poisons and of their an- 
tigenic properties. 

Another report of a similar nature was that coming from Jamie- 
son and Powell (1931) 676 who obtained from pneumococci toxic 
substances not unlike those which have been demonstrated by other 
workers as being produced by various streptococci. The action of 
the substances could be demonstrated by skin tests on human be- 
ings and on certain breeds of rabbits. By the same method, the se- 
rum of horses treated by subcutaneous injections of the toxic sub- 
stances could be shown to possess neutralizing properties for the 
alleged toxin. The neutralizing principle could be concentrated to 
a moderate degree in the refinement of globulins by the usual 
chemical procedures and, according to Jamieson and Powell, was 
independent of the small amount of protective antibodies present 
in the serum. 

HEMOTOXINS 

In 1927, Neill, Fleming, and Gaspari 958 described the results of 
studies on the antigenic or immunizing action of various prepara- 
tions of pneumococcal hemotoxin. The power of the reduced and of 
the reversibly oxidized forms of hemotoxin to evoke immunity were 
identical, but preparations in which the hemolytic principle had 
been rendered irreversible or destroyed by heat or by high con- 
centrations of hydrogen peroxide were inactive in this respect. The 
serum of rabbits and of horses injected with active hemotoxin neu- 
tralized the substance, while the antigenic action of the hemotoxin 
appeared to be independent of the protein fraction of the cell. 

Methods of Administering Antigens 

The manner in which Pneumococcus or its several antigens are 
introduced into the body of an animal of any given species, other 
factors being constant, may determine both qualitatively and quan- 
titatively the nature of the immunological response. In the early 



ANTIGENICITY OF PNEUMOCOCCUS 341 

studies on Pneumococcus one finds references to the subcutaneous 
injection of the organism or its products. The introduction of the 
intravenous route was followed by the more rapid appearance and 
a higher level of immunity in the treated animals. The administra- 
tion of antigens by way of the peritoneal cavity also offered ad- 
vantages over the subcutaneous route both in time of development 
of immunity and in heightened resistance. 

In 1922, Bronfenbrenner and Knights 149 published the results of 
a study on the comparative efficacy of the subcutaneous, intrave- 
nous, intratracheal, and intrapleural routes for the introduction 
of pneumococcal antigens. Rabbits were used and the immunity 
evoked was measured by the bactericidal, opsonizing, and protec- 
tive power of the serum following the different methods of treat- 
ment. The bactericidal titer of the serum from rabbits receiving 
intravenous and subcutaneous injections, as determined by the 
Heist and Solis-Cohen 634 technique, was approximately the same 
and was greater than that of the serum of animals injected either 
by the intrapleural or intratracheal routes. Both the intratra- 
cheal and intrapleural methods induced a much higher opsonic 
content of the immune serum than did the other procedures, while 
the administration of antigen by way of the blood, or into the tra- 
chea or pleura, caused the rabbits to yield serum containing pro- 
tective antibody, whereas the serum from rabbits treated subcu- 
taneously failed to confer passive protection on mice. 

In communications published in 1924, 192T, and 1930, Still- 
man 1330 " 2 ' 1334 ~ 5 supplied additional information concerning the 
nature and degree of the immune response in mice and rabbits re- 
sulting from inhalation, and from the subcutaneous, intramuscu- 
lar, and intravenous application of both living and dead pneumo- 
cocci. The serum of 80 per cent of rabbits injected intravenously 
with fixed amounts of heat-killed pneumococci and the serum of 60 
per cent of the animals injected intraperitoneally with similar 
doses contained agglutinins and all showed protective antibodies. 
Of rabbits receiving intramuscular injections, the serum of only 33 



342 BIOLOGY OF PNEUMOCOCCUS 

per cent contained agglutinins although 86 per cent were protec- 
tive. In the case of animals receiving antigen by the subcutaneous 
route, none produced agglutinins although protective substances 
could be demonstrated in 71 per cent of the individual serums. 

With skin sensitivity and the presence of type-specific agglu- 
tinins and protective substances in serum as criteria for the 
strength and specificity of pneumococcal antigens, Harley 
(1935) 592 obtained a higher degree of immunity and far greater 
type-specificity when vaccines of smooth and rough organisms 
were introduced intravenously than when injected intradermally. 

Killian (1924), 706 by continued subcutaneous and intraperi- 
toneal injections — the so-called bigeminal method — obtained a 
higher degree of protection in mice than by the subcutaneous route 
alone, and he believed that the fact argued against the acceptance 
of the hypothesis that a purely local immunity was necessary for 
establishing a state of protection against pneumococcal infection. 
By intravenous injection, extremely small quantities of antigen 
sufficed to produce protection, the immune state being demonstra- 
ble in an increasing degree from the third day after injection. 
Killian reported that a short refractory period ensued after the 
administration of antigen, and accordingly, delayed further anti- 
genic stimulation for four days until the period had passed. 

It is reasonable to assume that the duration of the refractory 
period — or the negative phase, as Wright termed it — depends on 
the route employed in the injection of antigen, the ability of the 
body cells to respond to subsequent stimulation being contingent 
upon the rate of absorption of antigenic material from the site of 
the injection. From foci under the skin, absorption would be less 
rapid than that from the peritoneal cavity and, quite obviously, 
the utilization of antigen would be most rapid when the injection is 
made directly into the blood stream. Because of the slower seep- 
age of antigenic materials from the subcutaneous tissues, that 
route is preferred for the production of antitoxin, but for the 



ANTIGENICITY OF PNEUMOCOCCUS 343 

elaboration of antibacterial substances, the intravenous method is 
the one of choice. 

INTRADERMAL INJECTION 

The intradermal injection of pneumococci and their products 
may be followed by the development of the immune state. Goodner 
(1928), 525 " 6 in studying the pathogenic action of living pneumo- 
cocci inoculated into the skin of rabbits, observed that the serum 
of animals surviving infection had acquired protective antibodies. 
Furthermore, within five days after a single intradermal injection 
of dead pneumococci, the normal rabbit developed resistance to in- 
fection. In two communications appearing in 1930, Julianelle 687 " 8 
reported that repeated injections of small doses of suspensions of 
heat-killed pneumococci of Types I and III into the skin of rab- 
bits induced species-specific but in no case type-specific antibodies 
for the organisms. However, animals developing only species-spe- 
cific antibodies after intradermal vaccination still possessed the 
ability to form type-specific immune substances when subsequently 
given intravenous injections of pneumococci of fixed types. De- 
spite the failure of heat-killed pneumococci when injected intra - 
dermally to evoke the formation of type-specific antibodies in the 
rabbit, devitalized cultures do, nevertheless, render the animal ac- 
tively immune to infection with pneumococci not only of homolo- 
gous but also of heterologous type. The injection into the skin of 
soluble derivatives of Pneumococcus in Julianelle's experiments 
was not followed by the production of active immunity. The de- 
struction or inactivation in the skin of rabbits of type-specific 
antigen injected intradermally was later observed by Harley 
(1935). 593 The somatic protein, on the contrary, appeared to be 
unaffected and stimulated the formation of species-specific anti- 
bodies. 

The observation of Julianelle was significant in that it revealed 
a hitherto unknown property of the epidermal cells of the rabbit. 



344 BIOLOGY OF PNEUMOCOCCUS 

The ability of that animal when injected intradermally to elabo- 
rate species-specific antibody, taken with the inability to manufac- 
ture type-specific antibody when similarly injected, points to a 
partial disruption of the molecular configuration of the pneumo- 
coccal antigen during its sojourn in the skin. The type-specific 
fraction of the antigen is probably destroyed and there remains 
only the somatic protein and carbohydrate of the bacterial cell to 
stimulate the immunological mechanism. That the mechanism is, 
nevertheless, fully capable of functioning in producing type-spe- 
cific immune substances is shown by the response of the rabbit 
when the antigen is introduced directly into the blood stream. The 
hypothesis is further supported by Julianelle's observation that 
the intradermal injection of heat-killed R forms as well as S forms 
of pneumococci calls forth the production of active immunity. 
Julianelle therefore demonstrated that the route of administration 
of pneumococcal antigen, at least in the rabbit, may be a deter- 
mining factor in the qualitative nature of the immune response. 

An analogous process taking place in the human body was re- 
ported in the same year by Francis and Tillett. 478 The repeated 
injection of the specific capsular polysaccharide of pneumococci of 
Types I, II, and III into the skin of patients suffering from lobar 
pneumonia was followed in the second or third week of convales- 
cence by the appearance of circulating antibodies for one or more 
heterologous types. However, in none of the normal controls was 
the phenomenon observed. In the next year (1931), Finland and 
Sutliff 446 " 7 confirmed the observations of Francis and Tillett. Pneu- 
monia patients receiving repeated intracutaneous injections of 
soluble specific substance from Type I, II, and III pneumococci, in 
a week or more developed in the blood agglutinins and protective 
antibodies sometimes heterologous for the infecting type of organ- 
ism but, unlike the effect in the rabbit, the immune bodies were ho- 
mologous for the type of capsular carbohydrate injected. 

In a subsequent report (1932), Finland and Sutliff 448 announced 
that the simultaneous intradermal injection into normal human be- 



ANTIGENICITY OF PNEUMOCOCCUS 345 

ings of specific polysaccharide of the first three pneumococcal 
types and of protein and autolysates derived from organisms of 
Types I and II was followed by the appearance or increase of the 
pneumococcidal power of whole defibrinated blood and in most in- 
stances by the presence of agglutinins and protective antibodies 
for one or more types of pneumococci. A single intradermal injec- 
tion of 0.01 milligram of the capsular polysaccharide of pneumo- 
cocci of Types I, II, or III, or four similar daily injections, re- 
sulted in the development of antibodies corresponding in type only 
to that of the antigen injected. A single injection into the skin of 
autolysates obtained from virulent Type I, II, and III pneumo- 
cocci gave rise to an increase in the pneumococcidal power of whole 
blood and the appearance of homologous type-agglutinins and 
protective antibodies in about one-third of the normal subjects. A 
similar injection of pneumococcal protein, on the contrary, failed 
to evoke specific antibodies to any appreciable degree. 

Further evidence of the antigenic action of specific polysaccha- 
rides injected into the skin of normal individuals was presented by 
Zozaya and Clark 1590 who, after the intradermal injection of solu- 
ble specific substance of pneumococci of Types I, II, and III were 
able to demonstrate homologous precipitins and protective anti- 
bodies in the serum of human beings, who gave no history of recent 
pneumococcal infection. Another observation of these authors may 
be repeated here. It was found that by increasing the surface area 
of the carbohydrate by adsorption on charcoal or celloidin parti- 
cles it was possible to evoke antigenic properties in preparations 
that otherwise appeared to lack immunizing action and to en- 
hance the action of preparations that already possessed these 
properties. Francis, 474 in a still more recent paper (1934), de- 
scribed experiments in which he studied the antigenic action of the 
original and the acetylated forms of the specific polysaccharide of 
Pneumococcus. Weekly doses of 0.01 milligram were administered 
intradermally over a period of three weeks to normal individuals. 
The experiment showed that both the original soluble specific sub- 



346 BIOLOGY OF PNEUMOCOCCUS 

stance and the acetylated polysaccharide of Pneuraococcus stimu- 
lated the development of type-specific agglutinins, precipitins, and 
mouse-protective antibodies in the serum of normal human beings. 
A consideration of the work of Julianelle, of Francis and Til- 
lett, of Finland and Sutliff, and of Zozaya and Clark demonstrates 
that the antigenic constituents of Pneumococcus introduced by the 
intradermal route may act as immunizing agents and that, while 
in man both type-specific immunity and corresponding type-spe- 
cific antibodies may be developed, the skin of the rabbit possesses 
the power so to alter pneumococcal antigen that only species-spe- 
cific immunity results. The difference in the epidermal and possibly 
the other somatic cells of animals of diverse species in their reac- 
tion to the parenteral introduction of Pneumococcus and its de- 
rivatives offers a field for study which should aid in clarifying some 
of the questions concerning the physiological processes involved in 
the development of the immune state. 

INHALATION AND INTRABRONCHIAL INSUFFLATION 

After giving repeated inhalations of Type I Pneumococcus, 
Stillman 1331 ' 2 demonstrated agglutinins and protective antibodies 
in the serum of rabbits so treated. Agglutinin production appeared 
to remain stationary after the fifth exposure to antigen, while the 
protective antibody content steadily rose. Repeated inhalations of 
killed pneumococci by mice resulted in only a slight degree of im- 
munity, but when living organisms were similarly administered a 
definite degree of active immunity could be induced in the animals. 

Eguchi (1925) 351 confirmed Stillman's observation on the pro- 
tection developing in mice following the inhalation of killed pneu- 
mococci, and found further that immunity could be induced by 
organisms of Type II as well as those of Type I. Maeji 858 also re- 
ported that, in the case of antivirus, inhalation was an effective 
mode of protecting young rabbits against subsequent infection 
with Pneumococcus, and that the degree of protection increased 



ANTIGENICITY OF PNEUMOCOCCUS 347 

with the quantity of inhaled antivirus and the number of applica- 
tions. 

Although Cooper (1926) 276 failed in attempts to immunize rab- 
bits to Pneumococcus by either the subcutaneous or intradermal 
injection of heat-killed cultures, he was able by applying the vac- 
cine beneath the buccal membrane of the cheek to protect the ma- 
jority of rabbits so treated for a period of four months against a 
fatal dose of the organism. Instillation of the vaccine into the eye 
or swabbing the nose and mouth with the agent failed to result in 
any effective immunity. Stuppy and Falk (1928) 1351 reinvesti- 
gated the subject presented by Cooper. Rabbits receiving ten to 
twenty daily injections of heat-killed Type I pneumococci into the 
buccal mucosa later survived inoculation with 1,000 M.L.D. of 
the same strain; of the animals receiving five preliminary injec- 
tions three out of four lived; while all rabbits given only one or 
two injections died. Ten daily subcutaneous, intradermal, intra- 
venous, and intratracheal injections or the administration of the 
vaccine by intratracheal insufflation or by intraocular instillation 
(contrary to the claims of Cooper) protected the animals against 
subsequent infection, although vaccination by insufflation or instil- 
lation was less reliable as an immunizing procedure than was the 
administration of the killed culture into the buccal submucosa or 
into the trachea. 

Stuppy, Cannon, and Falk 1850 rendered rabbits immune to intra- 
peritoneal infection with pneumococci of Types I and II by the 
daily insufflation into the nose and throat of suspensions of heat- 
killed cultures of homologous type, but failed in similar attempts 
with Type III organisms. In rabbits so treated, the insufflation of 
living, virulent pneumococci induced a proliferative and exudative 
type of reaction in the lung in which the macrophage was the pre- 
dominant cell, accompanied by considerable numbers of eosino- 
philes. This altered reactivity of the pulmonary tissues was inter- 
preted by the authors as affording evidence of a definite, localized 



348 BIOLOGY OF PNEUMOCOCCUS 

type of tissue immunity in the lung. Heterologous cultures of liv- 
ing pneumococci induced the local immune reaction but to a lesser 
degree. 

ORAL, ADMINISTRATION 

When pneumococci enter the body by way of the alimentary 
canal, their constituents may eventually reach the body cells re- 
sponsible for the creation of immune bodies. In 1925, Eguchi 351 re- 
ported that attempts to immunize mice by feeding them Type I 
pneumococci met with some success when young mice were used but 
with none in the case of adult mice. Kimura (1927), 710 neverthe- 
less, was able to protect a large percentage of adult mice against 
one hundred to one thousand lethal doses of Type I pneumococci 
by dropping into the mouths of the animals large amounts of heat- 
killed cocci of the corresponding type. A similar result attended 
Maeji's attempts to induce active immunity in mice by spraying 
the mouth with suspensions of pneumococci of Types I and II. 
When the effort was successful, there developed cross-protection, 
especially between Types I and II, with some protective action 
against Type III strains. Maeji 849 subsequently (1929) was able 
to immunize young mice, rats, and rabbits by the daily feeding of 
large quantities of broth cultures of highly virulent Type III or- 
ganisms. Although young rabbits could be rendered resistant to in- 
fection with Type III pneumococci by the oral administration of 
cultures of the corresponding type, no agglutinins could be de- 
tected in the serum of the animals. 

For the purpose of increasing the permeability of the intestinal 
mucosa for pneumococcal antigen, McDaniels (1931) 876 fed young 
white rats first with egg-white and then thirty minutes later with 
autolysates of Type I Pneumococcus. When tested after the lapse 
of five days by intraperitoneal inoculation with a virulent strain 
of Type I Pneumococcus, all the animals showed toleration of 
many lethal doses of culture, while the rats that had received a 
preliminary feeding with egg-white exhibited a greater resistance 



ANTIGENICITY OF PNEUMOCOCCUS 349 

to infection than did those that had not been so fed. In a second 
communication (1932), McDaniels 877 reported attempts to ascer- 
tain the details of the immunological mechanism operating in 
orally immunized rats. Normal control rats and rats previously 
injected with egg-white and Type I pneumococcal autolysates were 
inoculated intraperitoneally with one hundred fatal doses of a 
virulent, homologous strain of Pneumococcus. All the normal rats 
died within two to four days, while 96 per cent of the vaccinated 
animals survived. Although the leucocyte count was subject to 
wide fluctuations, the immunized rats, six hours after inoculation, 
always showed an appreciably higher white-cell count than did the 
unvaccinated controls. In none of the vaccinated rats which sur- 
vived infection could pneumococci be demonstrated in the blood, 
although pneumococcemia was present in all the animals that suc- 
cumbed. 

Ross (1931 ) 1188 described the comparative immunizing action of 
soluble specific substance and of whole or dissolved pneumococci 
when fed to rats. One feeding of Type I SSS sufficed to protect 
the test animals against intraperitoneal injection of virulent Type 
I organisms, and the increased type-specific resistance was com- 
parable, with slight quantitative differences, to that following the 
oral administration of intact or dissolved pneumococci. Ross failed 
to immunize mice by a similar procedure and attributed the failure 
to some cell constituent active in the mouth of mice. In other 
papers (1932), Ross 1189 " 90 reported that the capsular polysaccha- 
ride of Type II Pneumococcus was incapable, when introduced by 
the oral route, of increasing the resistance of rats to infection. 
The soluble specific substance of Type III organisms induced pro- 
tection in rats to organisms of the homologous type but the per- 
centage of animals so protected was less than in the case of 
animals receiving the whole or intact cell by mouth. When the spe- 
cific polysaccharide of Type I Pneumococcus was fed to rats, no 
immunity against Type II or Type III organisms was obtained, 
and the ingestion of SSS of Types II and III did not protect the 



350 BIOLOGY OF PNEUMOCOCCUS 

animals against Type I organisms. An observation of Ross on the 
fate of the ingested polysaccharide was that a very large propor- 
tion of the substance, when fed to rats, appeared in the feces. 

Further experiments on oral immunization against Pneumococ- 
cus, when applied to rabbits, were described in 1932 by Kolmer and 
Rule. 745 The antigens comprised a) pneumococci of Types I, II, 
and III cultivated in sterile milk for twenty-four hours and heated 
at 60° for one hour, b) twenty-four-hour broth cultures of the 
same types exposed for two hours to a N/15 concentration of hy- 
drochloric acid, and c) sedimented cocci from twenty-four-hour 
broth cultures subsequently treated with sodium taurocholate. The 
materials were administered daily for seven days through a stom- 
ach tube, and one week after the last treatment the rabbits were 
inoculated intratracheally with living broth cultures. While to 
Kolmer and Rule the acid-killed cocci, of the three kinds of prepa- 
rations used, appeared to engender the highest degree of immu- 
nity, the figures are not significant because of the lack of uni- 
formity of the results and the low virulence of the Type II and 
Type III cultures employed in testing the resistance of the rabbits. 

In a second report (1933), Kolmer and Rule 747 stated that the 
oral administration of acid-killed vaccines made from Type I, II, 
and III pneumococci, in doses representing 100 to 1,000 million or- 
ganisms per kilo of body weight, were effective in rendering rabbits 
immune to subsequent intratracheal infection with pneumococci of 
the homologous type. Although occasionally a single dose of 1,000 
million cocci produced active immunity against infection by Type 
I Pneumococcus, the best results were obtained with a minimum of 
five daily doses. The immunity thus induced was transient, de- 
creasing after one month's time and practically disappearing in 
six months. Vaccines introduced by way of the mouth were inferior 
in immunizing action to similar agents injected subcutaneously. 
Using monkeys as test animals, and employing vaccines of pneumo- 
cocci killed by tricresol and by hydrochloric acid, Kolmer and 



ANTIGENICITY OF PNEUMOCOCCUS 351 

Rule confirmed the superiority of the subcutaneous over the oral 
route of administration. 

Host Response 

As in all physiological processes there are periods in the devel- 
opment of an animal when the somatic cells function more actively 
in absorbing alien material and in creating and extruding immune 
substances into the circulating blood. This power may be lacking 
in very young animals, but it appears to increase as the animal 
grows and reaches a stage of full vigor, and then to decline as the 
metabolism of the body cells wanes. In addition to quantitative dif- 
ferences due to age in the antibody content of serum of rabbits 
immunized against Pneumococcus, Baumgartner (1934) 91 " 2 de- 
tected qualitative variations as well in the antibodies elaborated by 
young and older animals. The fact is sufficiently well established in 
immunological practice to require no detailed discussion. 

Antagonistic Action of Soluble Specific Substance 
In preceding chapters, mention was made of the aggressin-like 
action of soluble specific substance, of the zonal effect observed 
when this substance was used as an immunizing agent, and of its 
antagonistic behavior in reactions between immune serum and 
Pneumococcus or its derivatives. The soluble specific substance, al- 
though non-toxic in itself, can enhance the invasive power of pneu- 
mococci (Felton and Bailey 419 ). The phenomenon may be an addi- 
tive effect which, in turn, may be due to some interference with the 
natural defense mechanism of the body. Whatever the explanation 
of this property of specific polysaccharides, it is a fact to be reck- 
oned with in the production of active immunity, in testing the im- 
munity established by the administration of pneumococcal anti- 
gens, and in measuring the potency of antipneumococcic serum. 

Felton and Bailey demonstrated that the immunizing action of 
soluble specific substance operated within certain prescribed lim- 



352 BIOLOGY OF PNEUMOCOCCUS 

its. A definite minimum of the material was obviously necessary to 
incite the formation of antibodies, but when the dose was in- 
creased, a point was reached beyond which no immunity developed. 
Saito and Ulrich (1928) 1214 also observed this zone of antigenic 
effectiveness with a carbohydrate preparation in producing a state 
of protection in animals. A similar observation on the cellular car- 
bohydrate was reported by Wadsworth and Brown (1931). 1466 " 7 
There can be no doubt that pneumococcal antigen, particularly 
the capsular polysaccharide, in excess of a certain optimal amount 
hinders its own immunizing action. 

Another manifestation of the antagonistic action of the soluble 
specific substance is seen in the blocking of the reaction between 
pneumococcal antigen and homologous antibody. Felton and 
Bailey 419 drew attention to the inhibiting power of SSS frequently 
noted in precipitin and protection tests when amounts of antigen 
and serum greater than the optimum are used. The authors as- 
cribed the antagonism to the presence of precipitable residues in 
immune serum as well as to an excess of soluble specific substance 
in the antigen. The work of Felton and Bailey and that of Sickles 
(1927) 1277 point to at least one factor responsible for the inhibit- 
ing power of the soluble carbohydrate. Through its precipitating 
action, the polysaccharide removes some of the protective antibody 
from immune serum and at the same time (Sickles) interferes with 
phagocytosis, consequently robbing the serum of protective prop- 
erties. The observation recalls that of Sia (1927), 1268 who found 
that Pneumococcus could adsorb normal opsonin from the serum 
of animals naturally resistant to pneumococcal infection — an ob- 
servation later confirmed by Ward (1930) 1480 _1 for normal human 
whole blood, and who also, like Sia, noted that the action was type- 
specific. Ward suggested that the zone of inhibition was caused by 
the specific precipitate formed by the antiserum and homologous 
capsular polysaccharide, which interfered, perhaps mechanically, 
with the ingestion of the pneumococci by leucocytes. 

Enders and Wu (1934) 362 reported that the A carbohydrate 



ANTIGENICITY OF PNEUMOCOCCUS 353 

from Pneumococcus possessed a greater anti-opsonic action than 
the deacetylated polysaccharide. The opsonic titer of normal hu- 
man serum was practically nullified by the addition of the A carbo- 
hydrate, while in immune serum the A carbohydrate brought about 
a greater diminution in opsonic activity than did its derivatives. 
The result was therefore a complete inhibition of the pneumococ- 
cidal action of normal serum and a partial blocking of the same 
action of specific immune serum. The inhibitory or antagonistic be- 
havior of the carbohydrate fraction of Pneumococcus calls for 
careful discrimination in the selection of the antigenic dosage of. 
pneumococcal materials for the production of active immunity and 
also for conducting serological reactions which have to do with the 
immune state. 

Summary 

The substance of the material presented in this chapter may be 
expressed as follows : The immunizing properties and type-specific 
antigenic action of Pneumococcus and its derivatives are directly 
proportional to the virulence of the culture employed and appear 
to be the same whether the organism is in a living condition or ap- 
propriately killed by heat or by formalin, the integrity of the 
immunizing principle being better preserved in suspensions of heat- 
killed than of formalinized pneumococci. Filtrates from fluid cul- 
tures and watery or saline extracts of pneumococci, representing 
as they do only a part of the antigenic components of the pneumo- 
coccal cell, are more limited in immunizing properties than the en- 
tire cell. Although specially prepared broth cultures may exhibit 
a toxic action in animals, it does not necessarily follow that the in- 
jection of the alleged toxins results in the production of pneumo- 
coccal antitoxin. To be sure, some evidence of such a possibility 
has been presented but at the present time it would be going be- 
yond the facts to say that Pneumococcus contains or elaborates a 
true toxin or that antitoxic processes are a recognized part of the 
immunological mechanism against Pneumococcus. 



354- BIOLOGY OF PNEUMOCOCCUS 

Because of simplicity of preparation, stability, and freedom 
from undesirable secondary action, heat-killed vaccines are pref- 
erable to those prepared with bile, bile salts, soaps, or other sol- 
vents or devitalizing agents. Sensitized cocci present no advan- 
tages in immunizing properties over cells untreated with specific 
immune serum. 

The route by which antigens are introduced into the animal 
body affects both the kind and quantity of specific antibodies 
called forth. Injection into the venous system is the most effective 
method for inducing a high degree of immunity and strict type- 
specificity of the antibodies. Injection into the peritoneal cavity, 
into the muscles, or under the skin likewise leads to the elaboration 
of antibodies but, owing to the respectively slower absorption of 
antigen, there is a broadening of the specificity of the immunologi- 
cal response. Injection of pneumococcal substance into the skin 
stimulates the formation of antibodies but type-specificity may be 
sacrificed for species-specificity. The introduction of antigen into 
the bronchi by inhalation, insufflation, or injection, and also by 
enteric paths, is even less effective in raising the level of resistance 
or in evoking demonstrable immune substances in the circulating 
blood. 

The age of the animal treated with immunizing agents is another 
condition that determines the ability of animals to respond, at 
least in a quantitative way, to antigenic stimuli, while different 
species of animals display various physiological traits when in- 
jected parenterally with Pneumococcus or its derivatives. 

The antagonistic effect of excessive doses of pneumococcal mate- 
rial, particularly of the specific capsular polysaccharide, may 
again be emphasized, since the amount of the soluble specific sub- 
stance in a vaccine influences the development of the immune state 
or of immune substances. 

These, then, are the several factors which condition the immuno- 
logical behavior of animals toward substances of pneumococcal 
origin and which qualify any definition of the term antigen. 



CHAPTER XI 
ANTIBODIES TO PNEUMOCOCCUS 

The nature of the immune substances appearing after the injec- 
tion of Pneumococcus, its constituents, or products into the animal 
body ; the detection and estimation of specific antibodies in im- 
mune serum; with a brief discussion of the factors which operate 
in establishing immunity to Pneumococcus. 

The introduction into animals of pneumococci, their deriva- 
tives, or their several metabolic products generates a variety 
of antagonistic substances which may serve to protect the animal 
against subsequent infection with virulent members of this bac- 
terial species. In the present chapter there will be discussed the 
substances to be found in the circulating blood after active immu- 
nization and analogous substances normally present in animals, as 
well as changes in the somatic cells contributing to the immune 
state induced by parenteral injection of pneumococcal materials. 
The manifestations of the physiological functions aroused by 
the administration to animals of different species of pneumococci 
in one form or another are many and varied. Furthermore, the 
substances evolved in the immune processes are so closely related 
and so intimately mingled in immune serum that a discussion of 
any one of the specific substances necessarily involves that of other 
associated antibodies. However, in reviewing the investigations of 
earlier workers, one finds that the various immunological effects 
were discovered one by one, until the mosaic of pneumococcal im- 
munity — still incomplete, to be sure — has assumed a definite pat- 
tern which can now be recognized by improved serological and 
biochemical methods. 

Agglutinins 

The first tangible effect referable to an altered bodily state fol- 
lowing the injection of pneumococci was that of agglutinin for- 



356 BIOLOGY OF PNEUMOCOCCUS 

mation. Meager as was Metchnikoff's 894 description of the phe- 
nomenon, it was undoubtedly he who, in 1891, first observed the 
clumping of pneumococci in immune serum. Issaeff, 673 two years 
later, confirmed Metchnikoff's observation, and added that the se- 
rum of rabbits vaccinated with Pneumococcus when mixed with the 
organism, instead of becoming cloudy, developed a deposit. It is 
probable that in one of Mosny's 933 experiments (1892) agglutina- 
tion took place although he interpreted the result in a different way. 
The incubation of a culture of Pneumococcus in normal rabbit se- 
rum was attended by a diffuse clouding of the medium with a 
granular sediment appearing after twenty-four hours, while in im- 
mune serum the faint cloud disappeared after eight hours due very 
likely to the sedimentation of agglutinated cells. In 1896, Wash- 
bourn 1486 undoubtedly observed agglutination, for he described the 
sediment accumulating in immune rabbit serum twenty-four hours 
after the addition of pneumococci as consisting "of pneumococci 
staining well and grouped in masses." 

It was Bezancon and Griffon 108 " 10 who, in 1897, recognized the 
phenomenon as agglutination and compared the effect in the case 
of Pneumococcus to the Widal test for the diagnosis of typhoid 
infection. Although there was no difficulty in demonstrating the 
agglutinative properties of fluids collected from animals dying 
from pneumococcal infections, the results obtained with serum 
from patients suffering from similar infections were somewhat con- 
fusing. However, Bezancon and Griffon gave a correct interpreta- 
tion to their results in concluding that from the standpoint of 
agglutination there existed several races of pneumococci. In the 
first five cases of pneumonia studied, the organisms isolated ag- 
glutinated well in immune serum and these strains were referred to 
by the authors as vulgaire pneumococci. By the agglutination 
method, the French workers differentiated a strain with all the 
characteristics of Type III Pneumococcus and were therefore the 
first bacteriologists to recognize the applicability of the agglu- 
tination reaction to the differentiation of Pneumococcus from re- 



ANTIBODIES TO PNEUMOCOCCUS 357 

lated bacterial species and to the serological classification of pneu- 
mococci. 

In 1902, Neufeld 974 took exception to some of his predecessors 
who had studied agglutination of pneumococci with specific serum. 
He excluded any experiments in which only thread-like growths 
were observed, and contended that in none of the previous studies 
was the serum diluted, nor were the observations made soon enough 
after the organisms and immune serum were mixed. It was in this 
communication that Neufeld first described the Quellung effect 
which appeared when homologous immune serum was added to 
pneumococci. Neither normal rabbit nor normal human serum 
agglutinated pneumococci but normal beef serum often caused 
clumping of the organisms. Neufeld also noted that dead as well as 
living pneumococci were susceptible to the clumping action of im- 
mune serum, and that in the reaction the cocci were not dissolved 
nor were the living organisms killed. Neufeld met with difficulties in 
preserving the agglutinative titer of some samples of immune rab- 
bit serum — a difficulty later experienced by Hintze (1921 ) 647 and 
others with rabbit serum. More recently, Valentine, McGuire, 
Whitney, and Falk (1931) 1443 reported that the agglutination 
titer of dried antipneumococcic serum kept at room temperature 
decreased more rapidly than the mouse-protective titer. However, 
when the desiccated serum preparations were preserved at ice-box 
temperatures, the potency of the serum in this respect was not ap- 
preciably affected. 

AGGLUTININS IN THE BLOOD OF PNEUMONIA PATIENTS 

The serum of patients convalescing from pneumonia was in some 
cases found by Neufeld to contain agglutinins. Sometimes their ap- 
pearance was observed shortly before crisis although the aggluti- 
nating power of convalescent serum was highest on the day after 
crisis. The later observation was duplicated in the same year by 
Huber, 663 who also reported that the agglutinative properties of 
convalescent serum progressively diminished and completely dis- 



358 BIOLOGY OF PNEUMOCOCCUS 

appeared ten days after crisis. Similar results were obtained by 
Rosenow (1903), 1158 who found that when culture and serum came 
from the same patient agglutination was most pronounced, but in 
none of the serums from sixty-five cases of pneumonia when tested 
against twenty-five strains of Pneumococcus did agglutination fail 
to take place. Jehle (1903) 678 was able to demonstrate relatively 
high agglutinating power in the serum of all cases tested of croup- 
ous pneumonia terminating in crisis but, contrary to Huber and 
Rosenow, Jehle observed that only comparatively small amounts 
of agglutinin were demonstrable forty-eight hours after crisis and 
that the antibody had practically disappeared four days later. 

In a second communication, Rosenow (1904) 1159 reported addi- 
tional tests on the serum of pneumonia patients which confirmed 
his earlier results, and made the observation that pneumococci re- 
planted from the agglutinated mass in immune serum were viable 
as long as thirty days after being agglutinated. Wadsworth 1455 
substituted for the whole culture as antigen saline suspensions of 
pneumococci sedimented from twenty-four to thirty-six-hour-old 
broth cultures and carried out agglutination experiments with im- 
mune serum obtained from rabbits which had previously been in- 
jected with dead and later with living pneumococci. With serum 
from pneumonia patients in dilutions of 1 to 10 to 1 to 20 the anti- 
gen gave positive reactions in five or six hours. Wadsworth con- 
firmed Neufeld's negative results for normal rabbit and bullock se- 
rum, but found that normal human serum in a dilution of 1 to 10 
agglutinated pneumococci in less than eighteen hours. When the 
serum was diluted in a proportion of 1 to 30, no agglutination 
took place. In 1905, Longcope 824 described the sediment occurring 
when pneumococci were added to serum obtained from pneumonia 
patients, and also the accompanying swelling of the bacterial cap- 
sule, previously observed by Neufeld. 

In a study of the various characters of a large number of strains 
of pneumococci from a variety of human sources, Kindborg 
(1905) 713 obtained immune rabbit and sheep serum of high agglu- 



ANTIBODIES TO PNEUMOCOCCUS 359 

tinin titer, but concluded that agglutination was specific only for 
the strain used in the preparation of the serum employed in the 
test. In the same year, Collins, 270 after immunizing rabbits succes- 
sively with heat-killed and then with living broth cultures, tested 
the serum so prepared against some seventy strains of Pneumo- 
coccus. The serum of an animal immunized with one particular 
strain of Pneumococcus agglutinated only seven of the organisms 
when used in a dilution equalling that in which the homologous 
organism agglutinated. 

Collins concluded from her study that pneumococci by reason of 
their agglutinative properties exhibit a tendency to separate into 
numerous groups, and also that by the reaction of agglutination 
Pneumococcus mucosus forms a distinct and consistent variety. 
The conclusions were based on results obtained by the method of 
agglutinin-absorption, which indicated that the agglutinating sub- 
stances consist of specific and group agglutinins. Collins, appre- 
ciating that pneumococci showed marked differences in ability to 
undergo agglutination, and believing that there existed different 
types or groups as far as their agglutinative ability was con- 
cerned, was convinced that it was not possible to establish a defi- 
nite relationship between the agglutination reaction and other 
characters of pneumococci except in the case of Pneumococcus 
mucosus. 

Two other reports published before the serological classification 
of pneumococci was established are those of Panichi (1907), 1047 " 8 
and of Cotoni and Truche (1912). 284 The former author, after 
testing the agglutinative property of a strain of Pneumococcus at 
different stages of growth in bouillon when added to serum from 
rabbits, sheep, and asses previously injected with the same culture, 
concluded that agglutinability of the organism was greater during 
the process of growth than after the cocci had attained full de- 
velopment. Curative serums, including one prepared by Pane, did 
not necessarily contain agglutinins. The experimental data sug- 
gest that the serums employed were of low potency and, hence, the 



360 BIOLOGY OF PNEUMOCOCCUS 

results are not significant. Cotoni and Truche tested some thirty- 
one miscellaneous strains of pneumococci isolated from men, guinea 
pigs, rabbits, and horses against thirty-eight samples of serum 
from patients suffering from pneumococcal infections and against 
immune serum from horses and sheep, as well as normal serum, 
but the results of the agglutination reactions were confusing. For 
example, normal horse serum, under the conditions of the experi- 
ment, agglutinated the majority of the strains while normal sheep 
serum affected only a few of them. The action of the immune 
serums was indefinite, and Cotoni and Truche concluded that iden- 
tification of pneumococci on the basis of agglutinability was un- 
certain. 

The appearance of agglutinins in the blood during the course of 
lobar pneumonia was studied by Chickering (1914), 222 who found 
the antibody to be present in a large percentage of cases due to 
Groups I, II, and IV. In the most severe cases and in fatal cases, 
agglutinins could not be demonstrated nor could any be found, by 
the technique employed, in the blood of patients suffering from in- 
fection with Type III Pneumococcus. When agglutinins were de- 
monstrable they usually appeared at the time of crisis, whereas in 
some cases agglutinins were present for only one day, while in 
others they persisted for several weeks. In infections due to pneu- 
mococci of Types I and II the reaction was always specific for the 
type of organism causing the infection, while in infections due to 
organisms of Group IV the strain was agglutinated only by a se- 
rum strictly homologous for that strain. 

In an investigation of the antigen-antibody balance in lobar 
pneumonia, Blake (1918) 126 noted that prior to or coincident with 
the appearance of agglutinins, pneumococci disappeared from the 
blood. Patients who developed an excess of agglutinins over anti- 
gen invariably recovered ; those who showed a progressive increase 
in the excess of antigen without the development of demonstrable 
antibodies invariably died. Clough 238 confirmed Chickering's find- 
ings in demonstrating pneumococcal agglutinins in approximately 




Photographby Louis Schmidt Courtesy of the Rockefeller Institute for Medical Research 
FR. NEUFELD 



ANTIBODIES TO PNEUMOCOCCUS 361 

three-fourths of the cases of lobar pneumonia tested, and sub- 
stantiated Blake's observation that pneumonia patients who failed 
to develop demonstrable agglutinins succumbed to the infection. 

The agglutination curve of the blood of pneumonia patients re- 
ceiving antipneumococcic serum was employed by Cole (1917) 258 
to explain variations in the curative action of the serum. 

AGGLUTINATION AND SEROLOGICAL CLASSIFICATION 

In studies carried on by Neufeld and Haendel 991 during the 
years 1909 to 1912, it was learnt that agglutination of typical 
and atypical pneumococci corresponded to susceptibility to the 
protective action of specific immune serum. In 1913, Dochez and 
Gillespie, 322 by the methods of mouse protection and agglutina- 
tion, were able to separate strains of pneumococci isolated from 
cases of lobar pneumonia into four groups. The close agreement in 
the results demonstrated the value of the agglutination reaction 
in differentiating pneumococci, while the absence of cross-agglu- 
tination between the majority of representatives of the fourth 
group attested the heterogenicity of that class. Hanes 588 applied 
the method to cultures of Pneumococcus obtained from lobar pneu- 
monia patients and, while confirming the work of Dochez and Gil- 
lespie, encountered difficulties in the serological identification of 
Type III organisms. In attempts to demonstrate specific agglu- 
tinins in the serum of rabbits highly immunized to cultures of the 
mucoid variety of Pneumococcus, the results were uniformly nega- 
tive, but when the decapsulating method of Porges was used, in 
every instance the organisms agglutinated with homologous Type 
III serum, showed constant cross-agglutination within the group, 
and failed to react with immune serum for Types I and II and 
for streptococci. Hanes, therefore, suggested that henceforth the 
name Pneumococcus mucosus should be adopted for organisms of 
this group instead of the older designation, Streptococcus mu- 
cosus. With the aid of the Porges technique, Nicolle, Jouan, and 
Debains 1011 obtained agglutination with strains of Pneumococcus 



362 BIOLOGY OF PNEUMOCOCCUS 

that previously had failed to agglutinate with immune serum of 
the authors' preparation or with samples of serum from America. 
Gillespie (1914) 516 reported that strains of Type I and II pneu- 
mococci exhibited narrow zones of agglutination whereas organ- 
isms of the other types showed broad zones, and ascribed the 
variations to the differences in the hydrogen ion concentration of 
the cultures used and to the greater susceptibility of pneumococci 
of Types I and II to the inhibiting action of salts. Armstrong 
(1921— 1922) 19 ~ 20 placed greater reliance on the method of agglu- 
tinin-absorption than on simple agglutination in the classification 
of strains of pneumococci, since he believed that the latter tech- 
nique was not always sufficient for recognition of type. On the 
basis of agglutinability, Olmstead, 10278 Griffith, 558 and later Cooper 
and her associates 272 " 4 extended the serological classification of 
pneumococci to include the present thirty-two separate and spe- 
cific types. 

AGGLUTINABILITY OF PNEUMOCOCCAL VARIANTS 

The experiments of Stryker 1348 in 1916 demonstrated that the 
biological changes produced in pneumococci by growth in homolo- 
gous immune serum included alterations in agglutinative behavior. 
In 1923, Blake and Trask 129 found that a similar treatment of 
pneumococcal cultures resulted, along with loss of virulence, in 
constant and distinct changes in agglutinability, with respect to 
both the character of agglutination and the zone of optimal reac- 
tion. The changes appeared to be not a gradual alteration of all 
members of a culture, but to consist in a rapid and complete 
change in individual organisms. Three main variants developed 
after growth in immune serum, and all exhibited marked differ- 
ences in agglutinability. Analogous changes in the agglutinability 
of typical pneumococci after cultivation under unfavorable condi- 
tions were reported in the same year by Yoshioka. 1564 Cultivation 
on unsuitable media at 39° and long drying in the desiccator 



ANTIBODIES TO PNEUMOCOCCUS 363 

brought about a marked decrease in the agglutinative ability of 
the cultures in the presence of homologous serum, and caused the 
development of agglutinability with heterologous serum. The 
changes appeared irregularly and suddenly. Megrail and Ecker, 888 
on the contrary, claimed that Type I Pneumococcus in contact 
with Type I serum resisted the immunological change, as did the 
same organism when passed through a series of sterile abscesses 
artificially produced in mice and rats. 

As has already been explained in Chapter V, the dissociation of 
typical pneumococci into degraded variants is accompanied by a 
loss of type-specificity and the development of species-specificity 
in the agglutinative properties of the organism. When the de- 
generative process has not extended too far and the organism is 
then appropriately stimulated by animal passage with the accom- 
paniment of vaccines of typical strains or by growth in antirough 
serum, the organism may develop type-specific agglutinogens as it 
approaches the parent strain in its biological characters. 

AGGLUTININS IN THE BLOOD OF ANIMALS 

Bull (1915-1916) 170 ' 172 " 4 ascribed to agglutinins a decisive part 
in the resistance of animals to pneumococcal infection. He found 
that the injection, of small quantities of specific antipneumococcic 
serum brought about almost instantaneous and specific agglutina- 
tion of pneumococci in the circulation of infected rabbits. In all 
instances of both natural and passive immunity investigated, ag- 
glutination of the bacteria within the blood of the infected animal 
was followed by the rapid removal of the agglomerated cocci from 
the circulation and their destruction either by phagocytosis or in 
the capillary systems of the viscera. Unagglutinated pneumococci 
might remain in the blood stream and produce a progressive sep- 
ticemia. 

There could be found only one reference in the literature to the 
possible effect of an alien infection on agglutinins in the blood of 



364 BIOLOGY OF PNEUMOCOCCUS 

immunized animals. Reimann and Wu (1930) 1132 reported that ex- 
perimental typhus fever in guinea pigs vaccinated with pneumo- 
cocci had no influence on the pneumococcal agglutinins. 

ADDITIONAL DATA 

According to Goodner, 528 agglutinins are associated with the 
least soluble globulins — the euglobulin fraction — of antipneumo- 
coccic horse serum. No references could be found regarding the 
distribution of pneumococcal agglutinins in the protein portions 
of specific immune serum of other animals. In a study of the 
changes in bacterial volume as a result of specific agglutination, 
Jones and Little (1933) 681 " 2 stated that during specific agglutina- 
tion globulin from immune serum is deposited on the surface of 
the organism. The increase in volume of pneumococci might be 
ascribed to the interaction of cellular carbohydrate and immune 
globulin — possibly agglutinin — which results in a swelling, or 
Quellung to use the Neufeld term, of the cocci. 

In the mechanism of the type-specific agglutination of Pneu- 
mococcus, Francis 472 found that when the organisms were not 
present in sufficient numbers to absorb completely all the anti- 
bodies from immune serum, more antibody was bound by cellular 
carbohydrate than was required for the process of agglutination. 
The excess of antibody thus fixed could then unite with additional 
amounts of soluble specific substance when the polysaccharide was 
added in soluble form to the agglutinated material. When an ex- 
cess of free SSS was added to an agglutinated mass of antibody 
and pneumococci, the organisms were redispersed, and in the sus- 
pended state were again specifically agglutinable. Francis con- 
cluded that the reactive substance of the pneumococcal cell in 
type-specific agglutination is the capsular polysaccharide. The 
observation agrees with the observation of Avery and Goebel 
(1933) 46 that Type I acetyl polysaccharide in purified form ab- 
sorbs from Type I antipneumococcic serum all demonstrable type- 
specific agglutinins as well as precipitins and protective anti- 
bodies. 



ANTIBODIES TO PNEUMOCOCCUS 365 

Precipitins 

While investigating the massing of pneumococci in the presence 
of immune serum, Neufeld 974 dissolved the organisms in bile, added 
the clear solution to the specific serum which had agglutinated the 
culture, and noted that a particulate substance became micro- 
scopically visible within a quarter-hour. The aggregations grew 
and finally formed macroscopic masses of peculiar form and with 
a hyaline appearance. Neufeld thus showed the close relation that 
exists between specific agglutination and precipitation, and the ex- 
periments indicated that both phenomena were due to the same 
elements in the bacterial body and in the immune serum but, owing 
to the physical state of the antigenic substance, differed only in 
the manner of manifestation. In the next year (1903), Wads- 
worth, 1400 applying Neufeld's technique to saline suspensions of 
pneumococci and using normal rabbit bile for solution of the or- 
ganisms, corroborated Neufeld's observations. In order to elimi- 
nate an}- action of the bile, Wadsworth shook the centrifuged cul- 
tures with strong salt solution, brought the suspensions to the 
isotonic point, and filtered them. The filtrate precipitated with im- 
mune rabbit serum as in the case of the bile solutions of the cocci. 
The experiment showed that the substance precipitable by immune 
serum was a constituent of the normal pneumococcal cell and that 
it could be extracted by suitable solvents. Panichi (1907), 1047 by 
using filtrates of broth cultures of pneumococci, obtained precipi- 
tation with serum from rabbits, sheep, and asses previously im- 
munized with the same organism. The reaction in the different 
serums varied in degree. A marked reaction was characterized by 
an immediate opalescence; flakes soon separated, became larger, 
and settled to the bottom of the fluid in the form of a membrane 
which did not diffuse on shaking. In milder reactions the sediment 
was easily dispersed. 

In 1917, Dochez and Avery 321 discovered that the urine of ani- 
mals experimentally infected with Pneumocoecus and also the 
urine and blood serum of individuals ill with lobar pneumonia 



366 BIOLOGY OF PNEUMOCOCCUS 

showed the presence of a specifically precipitable substance in al- 
most every instance during some stage of the disease. Precipi- 
tinogen appeared as early as twelve hours after the initial chill 
and in some cases was demonstrable as late as five weeks follow- 
ing defervescence. The authors at the time presented data on the 
chemical nature of the soluble specific substance which showed that 
the substance which formed in the animal body during pneumo- 
coccal infection and passed through the kidneys into the urine 
was the complex carbohydrate which later was identified as the 
specific capsular polysaccharide. 

A year later, Quigley, 1114 in following the precipitin reaction 
during the course of lobar pneumonia due to pneumococci of 
Types I, II, III, and Type (Group) IV, found that when the urine 
was tested at intervals of two to three days, the reaction gradually 
increased in intensity during a period of three or four days, per- 
sisted from two to nineteen days, and then gradually disappeared. 
There seemed to be no regularity as to the period in the disease 
when the precipitinogen appeared in the urine, nor as to the 
length of time it persisted. Furthermore, its presence seemed to be 
independent of crisis. In the same year Blake 125 reported that, 
in nineteen carefully studied cases, there was a definite relation 
between the excretion of soluble pneumococcal antigen in the urine 
and the development of antibodies in the blood in lobar pneumonia. 
Precipitins were found only in clinically mild cases with negative 
blood cultures and with no antigen in the urine ; and these patients 
recovered. Blake stated that daily estimation of the concentra- 
tion of soluble antigen excreted in the urine, taken with the num- 
ber of cocci per cubic centimeter of blood, had great prognostic 
value in the individual case of lobar pneumonia. 

The type-specificity of the precipitin reaction was verified by 
Blake (1917), 123 who employed as precipitinogen the peritoneal 
exudate from mice infected intraperitoneally with pneumococci of 
Types I, II, and III, and Group IV. The method applied to a large 
number of strains yielded consistently positive and specific re- 



ANTIBODIES TO PNEUMOCOCCUS 367 

suits, the precipitate forming immediately without incubation 
when exudate and serum were of corresponding immunological 

type- 
There exists more than presumptive evidence that antipneumo- 
coccal precipitin and agglutinin are the same substance. In a 
quantitative study of the precipitation and agglutination reac- 
tions, Heidelberger and Kabat 014 obtained results which seem to 
prove the truth of the assumption. After removing from a com- 
bined Type I and II antipneumococcic serum the species-specific 
antibodies by absorption with somatic protein and the C Fraction, 
it developed that the application of the quantitative agglutina- 
tion method of Heidelberger and Kabat and the quantitative pre- 
cipitation method devised by Heidelberger, Sia, and Kendall 030 and 
by Heidelberger, Kendall, and Soo Hoo 028 yielded figures, within 
the limits of the accuracy of the methods, practically identical for 
anticarbohydrate precipitin and agglutinin. Heidelberger and 
Kabat stated that while this relation held for unconcentrated 
serum, in purified antibody solutions somewhat more agglutinin 
than precipitin was found, which they considered might be due to 
alteration of a portion of the antibody in the process of purifica- 
tion. The quantitative correspondence of type-specific anticarbo- 
hydrate agglutinin and precipitin argues for the immunological 
and chemical identity of the two immune substances and supports 
the unitarian theory of antibodies originally formulated by Zins- 



ANTIPROTEIN PRECIPITINS 

The somatic protein of Pneumococcus possesses the property 
of stimulating the production of precipitin and of reacting with 
the antibodies so formed. Since the protein is common to pneumo- 
cocci of all types and to degraded variants as well as to smooth, 
virulent forms of the organism, its precipitinogenic activity, as 
might be expected, is specific only for the species and not for type. 
These facts were reported by Avery and Morgan (1925), M who 



368 BIOLOGY OF PNEUMOCOCCUS 

found further that antiprotein serum failed to agglutinate type- 
specific strains of Pneumococcus or to react with the carbohy- 
drate derived from them. A somewhat analogous reaction was de- 
scribed in the same year by Jungeblut. 698 When alcoholic extracts 
of washed, sedimented pneumococci of Types I, II, and III were 
mixed with tincture of benzoin, added to specific immune serum, 
and incubated at 40°, flocculation took place. Antipneumococcic 
serums of the three types with homologous antigens gave floccula- 
tion of varying intensity. The reaction was strictly specific for the 
bacterial species and at the same time might be highly type-spe- 
cific. 

SOMATIC CARBOHYDRATE (c FRACTION) 

A phenomenon first discovered by Tillett and Francis 1409 and 
later investigated by Ash, 25 by Francis and Abernethy (1934), 475 
by Abernethy and Francis, 2 and by Abernethy 1 still awaits an ex- 
planation. 

It was found that the serum of individuals acutely ill with lobar 
pneumonia possesses the capacity of precipitating the somatic or 
C carbohydrate derived from pneumococci. It was further demon- 
strated that the precipitating action of patients' serum with the 
C Fraction is demonstrable in the early stages of pneumonia, 
sometimes within twenty-four hours of the onset, persists through- 
out the course of the active disease, or persists or recurs along 
with the development of complications, and disappears during con- 
valescence. 

In the report of the most recent experimental study available, 
Abernethy 1 stated that the serum of rabbits infected intrader- 
mally by the Goodner technique fails to precipitate the C carbo- 
hydrate, whereas the serum of monkeys (Macacus cynomolgos) 
given an intrabronchial infection with Type III Pneumococcus is 
capable of precipitating the somatic polysaccharide. The prop- 
erty is demonstrable within the first twenty-four hours following 
the experimental inoculation and persists for two or three days 



ANTIBODIES TO PNEUMOCOCCUS 369 

during the period of active infection. With the recovery of the 
animal the reactivity of the serum disappears almost as abruptly 
as it appears with the onset of the disease. Abernethy reports that 
the study demonstrates: 

. . . the variable response of two different hosts to the same bacterial 
agent. In one species, the monkey, pneumococcus infection was accom- 
panied by the demonstration of certain changes in the serum during the 
acute period of illness. In the other, the rabbit, these changes were not 
observed. These observations suggest that during infection in the mon- 
key either some newly formed substance or some alteration occurs in the 
serum which renders it inactive in precipitation tests with this particu- 
lar polysaccharide derived from Pneumococcus. Assuming this to be the 
case, then the failure to demonstrate the phenomenon in the rabbit 
might be explained either by the absence of or qualitative differences in 
the serum during infection. 

The phenomenon of precipitation of the somatic or C polysac- 
charide of Pneumococcus cannot be explained on the basis of any 
of our present orthodox conceptions of immunity. The property 
is not limited to the serum of individuals ill with pneumococcal in- 
fection, since it is also possessed by the serum of patients suffering 
from rheumatic fever, bacterial endocarditis, and lung abscess, 
but not from other febrile diseases. 

Furthermore, unlike other specific antibodies, the substance ca- 
pable of precipitating the C Fraction appears in the serum of the 
pneumonia patient within the first twenty-four hours of the dis- 
ease, and — again unlike any antibodies that we know — disappears 
from the serum with the beginning of recovery. Because of their 
importance in the body's defense against pneumococcal and other 
infections, the discovery of the chemical identity of the precipitat- 
ing substance in the serum of pneumonia patients and of the chemi- 
cal or physical processes involved in the particulation of the so- 
matic polysaccharide is awaited with keen interest. 

Jungeblut believed that lipoid substances extracted from Pneu- 
mococcus were responsible for the reaction but, as the author 
pointed out, the method of preparing the alcohol-soluble antigen 



370 BIOLOGY OF PNEUMOCOCCUS 

did not preclude the possibility that certain impurities, protein 
or carbohydrate in character, might have been carried over into 
the extract. The lack of sharpness in type-specific action would 
denote the participation of cellular protein in the reaction. 

A precipitin phenomenon described by Boor and Miller 138 in 
1931 is yet to be explained. Nucleoprotein and non-protein frac- 
tions prepared from strains of Gonococcus gave precipitates when 
added to antipneumococcic serum of Types I, II, and III. The 
antigens were active in high dilutions, and displayed even greater 
precipitating power in the presence of antimeningococcic serum. 

THE MECHANISM OF SPECIFIC PRECIPITATION 

Gay and Chickering, 508 " 9 in 1914, found that the addition of a 
water-clear extract of pneumococci to homologous antiserum pro- 
duces a voluminous precipitate which carries down with it agglu- 
tinins and the greater portion of protective antibodies. Subse- 
quently, Chickering 223 found the reaction to be type-specific. In 
the reaction, therefore, it was evident that the precipitinogen con- 
sisted of some soluble component of the pneumococcal cell while 
the specific precipitin, as later shown by Goodner, 528 was a sub- 
stance associated with the euglobulin of the horse serum employed. 

Soluble specific substance. Heidelberger and Avery, 606 in 1923, 
isolated the soluble specific substance from Type II pneumococci 
and announced that it consisted mainly of a carbohydrate and 
that in as high a dilution as 1 to 3,000,000 gave positive precipi- 
tation with homologous antiserum. According to the authors, the 
precipitinogen was apparently a polysaccharide constituting the 
capsular substance of Pneumococcus. This important conclusion 
was the first evidence of a possible connection between the capsular 
material and the specific relationships of pneumococci, and ac- 
counted for the type-specificity of the precipitin reaction. 

Felton and Bailey 420 fully substantiated the work of Gay and 
Chickering on the nature of the precipitate, and also the observa- 
tion of Heidelberger and Avery 607 that a single component of 



ANTIBODIES TO PNEUMOCOCCUS 371 

Pneumococcus — the soluble specific substance — was concerned in 
the particulation of type-specific antibody in immune serum. 

Heidelberger, Goebel, and Avery (1925) 613 confirmed the par- 
ticipation of the soluble specific substance in immunological reac- 
tions and refined the methods for the isolation of the polysaccha- 
ride. The purified substance from Type I Pneumococcus gave a 
specific precipitin reaction with homologous Type I antipneumo- 
coccic serum and could be detected in a dilution as great as 1 to 
6,000,000. Similar refined preparations from Type II and Type 
III pneumococci showed antigenic reactions of approximately the 
same magnitude, and in the precipitin reaction exhibited strict 
type-selectivity for the precipitins in homologous serum. The ob- 
servations substantiated the view that the polysaccharides were 
the actual specific antigenic substances of Pneumococcus. 

That the specific precipitinogen in Pneumococcus is evidently 
a soluble carbohydrate was shown in a different manner by Schie- 
mann and Casper (1927), 1228 who dissolved pneumococci in sodium 
taurocholate, and found that the solutions gave type-specific pre- 
cipitates with homologous immune serum. The authors rightly be- 
lieved that the soluble carbohydrate obtained by lysis of the cocci 
with the bile salt was the same as the type-specific polysaccharide 
isolated and tested by Avery and Heidelberger. 

Later (1931), Avery and Goebel 45 proved that the type-speci- 
ficity of the interaction of Pneumococcus and homologous immune 
serum was due to the capsular polysaccharide and not to the pro- 
tein of the cell. The serum of rabbits immunized with an artificial 
antigen, prepared by combining a specific derivative of the cap- 
sular polysaccharide of Type III Pneumococcus with globulin 
from horse serum, was found to contain specific precipitins for 
the Type III polysaccharide and, in addition, precipitins for horse 
globulin. It was, however, the capsular polysaccharide that was 
the determining factor in the specificity of the antigen as a whole. 
Subsequently, Avery and Goebel 46 proved that the acetyl poly- 
saccharide, besides being antigenic in the sense of being capable of 



372 BIOLOGY OF PNEUMOCOCCUS 

stimulating the production of specific antibodies, was also anti- 
genic in that, in a highly purified form, it reacted specifically with 
homologous precipitin and completely removed precipitin from 
immune serum. 

Balance between precipitinogen and precipitin.Morga.n (1923) 912 
demonstrated that a proper balance between antigen and anti- 
body was required to bring about the phenomenon of precipita- 
tion. In order to obtain the maximal precipitate from any given 
quantity of antipneumococcic serum, a definite or optimal amount 
of precipitinogen or soluble specific substance was necessary. The 
ratio between the quantity of immune serum and reacting sub- 
stance was found to be practically constant. Therefore, when an 
excess of precipitinogen was present, the resulting precipitate 
was not only decreased in amount, as determined by inspection and 
weight, but its appearance was altered. The importance of the 
relative proportions of antigen and antibody was also demon- 
strated by Dean and Webb (1926) 311 who, employing normal 
horse serum and its homologous antiserum, devised a method by 
which it was possible to determine quantitatively the amount of 
antigen and antibody and therefore the optimal ratio between the 
two components necessary for complete precipitation. 

Precipitin index. Sobotka and Friedlander (1928) 1299 deter- 
mined that the sensitivity of the precipitin reaction with anti- 
pneumococcic serum could be expressed by the product of the con- 
centrations of the two reacting substances, antigen and antibody, 
and defined the precipitin index (P.I.) as one-millionth of the re- 
ciprocal value of the product. In investigating the zonal phenome- 
non and its bearing on the absolute concentration and equivalent 
weight of antibody, the authors found that the greater tendency 
toward the exhibition of a post-zone in Type III precipitation 
was connected with the lower acid equivalent of the homologous 
capsular polysaccharide. The addition of normal serum as well as 
an increase in pH promoted the post-zonal effect. By determining 
the precipitin index, it was possible to recognize and to eliminate 



ANTIBODIES TO PNEUMOCOCCUS 373 

zonal irregularities. Smith 1298 later (1932) applied the principle 
of optimal proportions to precipitin tests with Type I soluble 
specific substance and homologous antibody and reported that the 
method gave a true index of the protective power of the serum. 
More recently Felton devised a test for the combining power of 
antipneumococcic serum with specific polysaccharide. 

In the description by Francis, 472 already cited in the discussion 
of agglutinins, it appeared that differences in the ratio of antigen 
and antibody which affect the agglutination reaction apply also 
to the precipitation reaction. When a solution of soluble specific 
substance was added in excess to homologous immune serum, a 
pro-zone was created in which precipitation was inhibited. As in 
the agglutination reaction, Francis showed that it was the cap- 
sular polysaccharide which constituted the precipitinogen. 

Quantitative relations in precipitation. In 1929, Heidelberger 
and Kendall 616 published the first of a series of communications on 
a quantitative study of the precipitin reaction as related to sol- 
uble specific substance and homologous immune serum. After pre- 
cipitating Type III antipneumococcic serum with Type III poly- 
saccharide, the authors measured the amount of nitrogen in the 
supernatant fluid of the precipitated mixture, and then from the 
nitrogen content calculated the amount of protein remaining in 
solution. The authors then tested the experimental data by the 
law of mass action to determine whether the reaction showed 
analogies to the behavior of simpler ionic reactions. Depending on 
the relative amounts of the reactants, the specific precipitate ap- 
peared to be a mixture of 'varying proportions of two compounds, 
or a whole series of compounds, containing hapten and antibody 
in varying proportions, whose limits could be expressed by the 
following equilibriums, A and S being respectively equivalent 
amounts of antibody and Type II soluble specific substance : 

(1) A + S^± AS (120:1 ratio) 

(2) AS + S ^± AS 2 (60: 1 ratio) 



374 BIOLOGY OF PNEUMOCOCCUS 

According- to Heidelberger and Kendall, an inhibition-zone effect 
appears to be a chemical equilibrium which might be expressed by 

(3) AS 2 + S^±AS 3 

In equilibrium (1) the reaction tends to proceed strongly to the 
right since AS is only slightly soluble. Reaction (2) will go far- 
ther to completion and more AS 2 will be precipitated (since AS 2 
has an appreciable solubility and dissociation tendency) when a 
little S is added after equilibrium (2) has been reached. When 
much S is added, equilibrium (3) comes into play and the precipi- 
tate dissolves. The authors suggested as an analogy the reaction 
between silver and the cyanide ion, where a small amount of CN" 
causes a precipitate of AgCN, and an excess of CN" causes the 
formation of the silver cyanide complex which is soluble. 

Heidelberger, Sia, and Kendall (1930) 630 described a rapid and 
simple method for the approximate determination of the specifi- 
cally precipitable protein in Type I antipneumococcic serum. In- 
asmuch as a close parallel was found to exist between the amount 
of specifically precipitable protein and the number of mouse pro- 
tection units, and because of the rapidity, simplicity, and economy 
of the method, the authors proposed its use instead of the mouse 
protection test as a basis for the titration of standard serum and 
for the comparison of other serums with the standard. 

Heidelberger and Kendall (1932) 619 devised a method, based on 
the precipitin reaction, for the microdetermination of the spe- 
cific polysaccharide of Type III Pneumococcus. By the method, 
as little as 0.01 milligram of Type III polysaccharide could be 
measured, while the procedure appeared to be applicable to any 
specific polysaccharide upon standardization of an homologous 
antiserum or antibody solution in the region of excess antibody. 
In another paper, 620 the authors described precipitin reactions 
performed with partial hydrolytic products of the specific cap- 
sular polysaccharide of Type III Pneumococcus freed quantita- 
tively from unhydrolyzed specific polysaccharide. The fractions 



ANTIBODIES TO PNEUMOCOCCUS 375 

yielded specific precipitates, in dilutions of 1 to 1,000,000, with 
Type III antipneumococcic horse serum, but failed to precipitate 
homologous rabbit antiserum, giving rise to specific inhibition. 
The authors found further that aldobionic acid, the structural 
unit of Type III polysaccharide, did not precipitate homologous 
antiserum. 

With Soo Hoo, Heidelberger and Kendall (1933) 628 described 
a method, developed from the previous work of the two last-named 
authors, for the microestimation of precipitin in antiserum. The 
technique involved the use of a deep-red protein dye, the R salt of 
azo-benzidene-azo-crystalline egg albumen. The procedure gave 
the actual weight of precipitin and could be applied to the maxi- 
mal amount of precipitable antibody in any antiserum. The ob 
servation that four samples of immune rabbit serum produced in 
response to injection of the dye contained over one hundred times 
as much precipitin as the antigen injected, appeared to the au- 
thors as supplementing the growing mass of evidence against the 
theory that specific antigen fragments are actually incorporated 
into the antibody molecule. 

In 1935, Heidelberger and Kendall 622 pointed out that the usual 
immunological technique, namely, incubation of precipitin reac- 
tions at 37° for two hours and allowing the tubes to stand in the 
ice-box over night, while resulting in the maximal precipitation of 
antibody from immune rabbit serum, in the case of antipneumo- 
coccic horse serum or purified antibody, does not permit the estab- 
lishment of a true equilibrium or the precipitation of the maximal 
amount of antibody nitrogen. The authors recommended that 
analyses of antipneumococcic horse serum should therefore be car- 
ried out at 0° and the tubes containing the antigen and immune 
serum should be allowed to stand in the cold for at least twenty- 
four hours in order to ensure the completion of the reaction. 

In an accompanying paper, Heidelberger and Kendall 623 re- 
ported the results of additional study of the quantitative relations 
in the precipitin reaction between Type III polysaccharide and 



376 BIOLOGY OF PNEUMOCOCCUS 

homologous immune horse serum, which led the authors to the con- 
clusion that the reaction could be accounted for quantitatively by 
assuming the chemical combination of the components in a bimo- 
lecular reaction, followed by a series of competing bimolecular 
reactions which depend on the relative proportions of the com- 
ponents. From the data, Heidelberger and Kendall then developed 
additional mathematical formulas representing the developments 
in the mechanism of the precipitin reaction. As the authors re- 
marked in their 1929 communication, "Of all the reactions of im- 
munity the precipitin test is perhaps the most dramatic and strik- 
ing; while other immune reactions are more delicate, the precipitin 
test is among the most specific and least subject to errors and 
technical difficulties" ; and "The isolation of bacterial polysac- 
charides which precipitate antisera specifically and possess powers 
of haptens has not only afforded one of the components of a pre- 
cipitin reaction in a state of comparative purity, but has greatly 
simplified the analytical problem." By these exact quantitative 
methods, the precipitin reaction has conferred inestimable benefits 
on the immunologist in understanding the nature of immunological 
phenomena, such as the relations between antigen and antibody, 
in judging the purity of antigen, and in estimating the antibody 
content of antipneumococcic serum. 

The chemical study of capsular polysaccharides and pneumo- 
coccal antibody is furnishing clues to the process which operate in 
the union between type-specific antigen and homologous antibody. 
By simple chemical experiments, Chow and Goebel 226 showed that 
the immunological activity of pneumococcal antibody protein is 
to a great extent dependent upon the presence of free amino 
groups in the protein molecule. With the work of Landsteiner and 
van der Scheer 782 " 3 in mind and reasoning from the results of the 
study on synthetic antigens made by Avery and Goebel (1929), 44 
Chow and Goebel assumed that in the case of the type-specific anti- 
body of Pneumococcus the spatial arrangements of the polar 



ANTIBODIES TO PNEUMOCOCCUS 377 

groups in the immune protein may determine its specific capacity 
to react with the polysaccharide of the homologous type. 

In the carbohydrate of Type I Pneumococcus the authors be- 
lieved, on the basis of the experimental evidence, that the carboxyl 
groups of the polysaccharide are the dominant groups which in- 
teract to form the immune precipitate. The questions whether the 
carboxyl groups of the polysaccharide actually combine with the 
amino groups of the antibody protein and whether the formation 
of an insoluble precipitate involves further chemical change in the 
protein molecule, such as specific denaturation, cannot be an- 
swered at present. However, Chow and Goebel concluded : 

The specificity of this reaction is determined by the stereochemical 
relationship of the dominant polar groups in the reacting molecules, 
whether they be antigen or antibody. If the spatial pattern of the polar 
groups of both antigen and antibody is of exactly the correct order, 
then union occurs. If, however, this relationship is disturbed by arti- 
ficial means, as has been experimentally demonstrated by covering the 
dominant polar group of either polysaccharide or antibody with a 
chemical radical, the pattern is destroyed and union between them is 
either greatly modified or fails to take place. When the original consti- 
tution of the reacting substances is restored, however, serological speci- 
ficity is regained. 

CORRELATION OF PRECIPITINS WITH OTHER ANTIBODIES 

Many observations have been made on the quantitative relation- 
ships existing between precipitins and other specific antibodies in 
antipneumococcic serum. The discovery that the capsular poly- 
saccharide functions as the precipitinogen in the reaction, and the 
development of methods for evaluating the strength of antigen 
and antibody, have made possible more exact determination of the 
amount of precipitin in a given serum in relation to the content 
of other immune substances. Friedlander, Sobotka, and Banzhaf 
(1928) 493 estimated, under known conditions, the precipitin in- 
dices of a number of monovalent and polyvalent antipneumococcic 



378 BIOLOGY OF PNEUMOCOCCUS 

serums and found the indices to vary as did the number of pro- 
tective units. The ratio of the precipitin index to protective units 
in monovalent serums was between 2.8 and 4.8 for Type I and 
about ten times greater for Type III. Lower values prevailed in 
polyvalent antipneumococcic horse serum and in mixtures of 
heterologous monovalent serums. A relative increase in precipitin 
activity was found in the refined and concentrated serums tested. 

The experiments of Avery and Goebel (1931 ) 45 with the arti- 
ficially conjugated Type III capsular polysaccharide-horse serum 
globulin would seem to argue for the unity of type-specific pre- 
cipitins, agglutinins, and protective antibodies. 

In a systematic study of the quantitative relations existing be- 
tween the various specific antibodies in Type I and II antipneu- 
mococcic horse serum, Felton (1931) 407 determined for Type I 
Pneumococcus the correlation coefficient between protective and 
precipitin titer as 0.93 ; between protection and agglutination the 
figure was 0.80 ; between protection and neutralization it was 
0.88 ; and between protection and the amount of protein precipi- 
tated with specific carbohydrate it was 0.91. From this degree of 
correlation it appeared to Felton that, at least for freshly drawn 
horse serum, the precipitin test could be used to estimate the prob- 
able therapeutic value of antipneumococcic serum. 

The qualification made by Felton in respect to the freshness of 
the serum sample as influencing precipitation and protective titer 
was confirmed by Valentine, McGuire, Whitney, and Falk (1931 ) 1443 
in experiments on the effect of ageing on antibodies in dried anti- 
pneumococcic serum. At room temperature, agglutinin and pre- 
cipitin titer decreased to a greater degree than did the number of 
mouse-protective units, although the deterioration of the two first- 
named antibodies failed to take place at ice-box temperatures. 

In a more recent publication (1936), Barnes, Clarke, and 
Wight 82 compared the unit value of serums obtained by mouse 
protection with a) the water test; b) agglutination test; c) the 
authors' routine precipitation test; d) the optimal proportions 



ANTIBODIES TO PNEUMOCOCCUS 



379 



precipitation test ; e) Felton's test for combining equivalents ; and 
f) a modified precipitation test devised by the authors. The fol- 
lowing table taken from page 131 of the authors' communication 
shows the correlations found between precipitation, combining 
equivalents, agglutination, and mouse protection. 



Method of titration 


Serums 

tested with 

control 


Incubation 


End-point 
= highest 
dilution 
showing 


Antigen 


Correlation 
with mouse 




Pll 


P 403 


Hours 

at 40° 


Ice-box 


protection 


Agglutination .... 

Optimal propor- 
tions precipita- 
tion 


32 


50 

50 

50 




18 

50 






50 


2 

• 
2 

2 

• 




Over- 
night 

* 

Over- 
night 

Over- 
night 

• 

Over- 
night 


Trace of 
aggluti- 
nation 

• 

Definite 
floccu- 
lation 

Definite 
floccu- 
lation 

No un- 
com- 
bined 

SSS 

* 


Live 
broth 
culture 

SSS 

SSS 

1:10,000 
dilution 

SSS 

1:10,000 
dilution 

SSS 

1 : 10,000 
dilution 

• 


0.713 ±0.047 
0.909 +0.016 


Routine precipita- 
tion 


0.866 +0.024 


Modified routine 
precipitation . . . 

Combining equiva- 
lent 


0.930 ±0.012 
0.925 +0.014 




0.652 ±0.055 



Not applicable. 



By closely spacing the serum dilutions in the modified test, 
Barnes, Clarke, and Wight succeeded in obtaining precipitin titers 
which agreed more closely with estimations of the mouse-protec- 
tive value than with any of the other methods tested. It would 
seem, therefore, that the precipitin content, as determined by this 
method, is in close agreement with the amount of protective anti- 
body in specific antipneumococcic serum. 



380 BIOLOGY OF PNEUMOCOCCUS 

Complement-Fixing Antibodies 

The reaction of complement fixation is so far inferior in deli- 
cacy to that of agglutination, precipitation, or mouse protection 
for the serological diagnosis of pneumococcal types that the sub- 
ject can be dismissed with brief mention. Hanes (1914), 588 using 
the method, found that strains of Pneumococcus mucosus were 
more closely related to Pneumococcus than to Streptococcus. 
There was a certain amount of cross-fixation between immune 
serum of Types I and II, and Type III antigens, whereas anti- 
streptococcic serums deviated complement only in the presence of 
antigens made from homologous strains. Christensen (1922) 2289 
employed the method of complement fixation to check type-deter- 
mination of pneumococci of the first four types made by the ag- 
glutination reaction but, because the first-named method was more 
complicated and required a longer time for its execution, Chris- 
tensen did not recommend its use in place of agglutination. In the 
studies made by Avery and Heidelberger (1925) 49 on the anti- 
bodies demonstrable in the serum of rabbits immunized by injec- 
tion of intact pneumococci and of their carbohydrate and protein 
derivatives, the method of complement fixation yielded results 
agreeing with those obtained by specific precipitation. Bull and 
McKee (1929) 180 applied the method to test the production of 
antibodies in rabbits immunized with heat-killed broth cultures of 
pneumococci. Serum from animals immunized with a Type II 
strain fixed complement in the presence of antigens of Types I, II, 
and III, but the titer was always higher with the homologous 
antigen. The same observation held true for Type III antiserum. 
From rabbits injected with a Type (Group) IV organism, the 
serum was potent when the homologous organism was used as anti- 
gen and only slightly weaker in the presence of Type I antigen. Al- 
though the serums were low in agglutinin titer, their action was 
type-specific. 

The mechanism of the complement-fixation reaction with the 
components of Pneumococcus, and the disparity between the abil- 



ANTIBODIES TO' PNEUMOCOCCUS 381 

ity of specific antipneumococcic serums produced respectively in 
the rabbit and horse to bind complement in the presence of homolo- 
gous antigen, have been studied by Goodner and Horsfall. 538 With- 
out retailing the experimental details, it may suffice to abstract 
the discussion and summary. The experiments reported supported 
the view that the failure to obtain complement fixation with com- 
binations of pneumococcal capsular polysaccharide and specific 
immune horse serum is not due to some heterologous inhibitor in 
immune horse serum but is to be referred rather to some property 
of the horse antibody itself or some property of the immune ag- 
gregate resulting from the union of this antibody and the poly- 
saccharide. This property is lacking in the specific antibody in 
immune rabbit serum which, in the presence of homologous poly- 
saccharide, is capable under proper conditions of binding com- 
plement. Furthermore, the results support the view that the fixa- 
tion of complement is a phenomenon of selective absorption. That 
one type of aggregate absorbs complement while another fails to 
do so is curious, but far from unique. Goodner and Horsfall found 
a close parallelism in the fact that horse antibody-polysaccharide 
aggregates absorb cephalin, while aggregates containing rabbit 
antibody selectively absorb lecithin. 

The essential role of serum lipids in the demonstration of the 
phenomena of specific precipitation and agglutination has been 
described by Horsfall and Goodner. 657 The removal of lecithin 
from antipneumococcic horse serum and, to a lesser degree, the ex- 
traction of cephalin from antipneumococcic rabbit serum cause a 
loss of the visible phenomena of agglutination and precipitation. 
It was found that initial activity of type-specific antibody can be 
restored to extracted immune horse serum by the addition of leci- 
thin, and to extracted rabbit serum by the addition of cephalin. It 
is therefore probable that the content of these two phospho-lipids* 
in the serum of the two animal species may, in part, account for 

* In a recent brief communication, Horsfall, Goodner, and MacLeod659 de- 
scribe the antibody in pneumococcal immune horse serum as a lecithoprotein 
and that in immune rabbit serum as a cephaloprotein. 



382 BIOLOGY OF PNEUMOCOCCUS 

the differences in the immunological behavior of immune horse and 
rabbit serum. 

Inasmuch as complement fixation does not occur in the absence 
of particulation and since particulation is a secondary phenome- 
non in the reaction, the authors regard complement fixation as a 
tertiary manifestation. 

The evidence presented in the communications cited and in other 
reports is sufficient to demonstrate that the method of complement 
fixation offers no advantages over other serological methods for 
the identification of pneumococci or of types within the species. 

Bactericidins 
With the advancement of bacteriological knowledge, the pneu- 
mococcidal action of both normal and immune serum has been 
found to be a more complicated process than it was earlier con- 
ceived to be. Instead of being solely a function of possible bac- 
tericidins in serum, the destructive effect of blood involves the ac- 
tion not only of serum but of leucocytes and of fixed tissue cells. 
Since the pneumococcidal power of blood depends on the asso- 
ciated participation of tropins or opsonins and phagocytes, the 
special features of their combined action will be described later in 
the present chapter, while the nature of other inherent defenses of 
the animal body will be discussed in a subsequent chapter. 

Antihemotoxin 
Experiments on the antigenic action of pneumococcal hemo- 
toxin reported in 1914 by Cole 252 indicated that the serum of rab- 
bits and sheep immunized with hemolytic extracts of Pneumococ- 
cus had acquired increased power to inhibit the lytic effect of the 
extract on erythrocytes. In studies on the oxidation and reduction 
of immunological substances, Neill 952 and his colleagues 958 " 9 com- 
pared the immunological response to the injection into rabbits of 
reduced and oxidized pneumococcal extracts, and succeeded in 
producing a neutralizing antibody by immunizing the animal with 



ANTIBODIES TO PNEUMOCOCCUS 383 

the hemolytically inactive hemotoxin present in oxidized solutions 
as well as with the active hemotoxin. The antibody appeared to be 
a species-specific antihemotoxin neutralizing the hemotoxin from 
all types of pneumococci, since it was without effect on the hemo- 
toxins of tetanus and Welch bacilli. Neill with Fleming and Gas- 
pari determined that for the production of antihemotoxin in the 
rabbit or horse it was essential that the hemotoxic antigen be used 
in an unheated condition. 

A similar antihemotoxic serum was developed by Cotoni and 
Chambrin (1928) 282 in rabbits, sheep, and horses after immuniz- 
ing the animals with living cultures, extracts of living and dried 
pneumococci, as well as with dried organisms killed by alcohol and 
ether. The antihemotoxin was stable after heating for one-half 
hour and, as Neill 950 found, was devoid of type-specificity but pos- 
sessed species-specificity, because when tested against the hemo- 
lysins of streptococci, tetanus bacilli, septic vibrios, and Bacillus 
perfringens, the serum displayed no neutralizing action. Doubt 
was cast on the species-specificity of antihemotoxins by Todd 
(1934-), 1412 who apparently was able to neutralize, to a limited ex- 
tent, the hemolysins of Type II and Type III pneumococci with 
antistreptolytic serum. The degree of neutralization was not neces- 
sarily correlated with the antistreptolytic titer, and the different 
hemolysins could be distinguished by quantitative serological 
methods. The partial antigenic overlapping of hemolysins could 
only be demonstrated by the use of hyperimmune serum. 

Antitoxin 

Boehncke and Mouriz-Riesgo (1915), 134 notwithstanding the 
fact that they were never able to obtain any very active pneumo- 
coccal toxin, believed that the curative action of some of the im- 
mune serum prepared by them was due to its antitoxic qualities. 
Olson (1926) 102930 claimed that it was possible to immunize mice 
by serial injections of pneumococcal toxin prepared by allowing 
sodium ricinoleate to act on pneumococci, but the author pre- 



384 BIOLOGY OF PNEUMOCOCCUS 

sented no serological evidence of the presence of antitoxin in the 
serum of the immune animals. In addition, serum from normal 
horses, sheep, rabbits, and chickens, as well as antipneumococcic 
serum or pneumococcic antibody solution, was found to possess 
little power to prevent the lung changes or cutaneous reactions 
evoked in mice by the toxin. 

Clowes, Jamieson, and Olson (1926), 244 by injecting rabbits, 
sheep, and horses with progressively increasing doses of sterile 
toxic extracts prepared by the sodium ricinoleate method of Lar- 
son, obtained a serum that neutralized the skin-reacting substance 
contained in the extracts, and suggested as a provisional unit of 
antitoxin the amount of serum required to neutralize one million 
skin-test doses of toxin. Concentration of the immune serum ef- 
fected the removal of 99.9 per cent of the total serum-protein 
without appreciable loss of antitoxin. Larson, 788 in the same year, 
administered an immune serum prepared and tested in the same 
manner to patients ill with lobar pneumonia and, because the pa- 
tients showed a rapid drop in temperature and experienced relief 
of subjective symptoms following the administration of the serum, 
he believed that the serum actually contained antitoxin. 

The work of Parker (1929), 1061 referred to earlier in the text, 
has a definite bearing on the question of the possible existence of 
pneumococcal antitoxin. Serum prepared by the author in rab- 
bits and horses by using sterile filtrates of pneumotoxin as anti- 
gens, under certain conditions, protected guinea pigs against the 
pneumonia caused by the intratracheal injection of living pneu- 
mococci and toxic pneumococcal autolysates. The protection thus 
conferred was heterologous for type and appeared to be due to 
some immune substance other than protective antibodies, since the 
latter could not be demonstrated in the active serum of rabbits 
and horses immunized with sterile filtrates of toxic autolysates of 
Pneumococcus. In a second publication, Parker and McCoy 
(1929) 1062 described a method for standardizing the potency of 
antitoxic horse serum and set as the unit of toxin the amount of 



ANTIBODIES TO PNEUMOCOCCUS 385 

filtered autolysate which, when injected intratracheally, would 
kill a guinea pig weighing 200 to 210 grams in from four to 
twenty-four hours with typical symptoms and necropsy findings ; 
while one unit of antitoxin represented the smallest amount of 
serum necessary to protect a guinea pig of the same weight 
against one unit of toxin when the test toxin-serum mixture was 
injected intratracheally. Neither normal horse serum nor anti- 
pneumococcic horse serum containing 500 protective units per 
cubic centimeter when used in a 1 to 10 dilution exerted a detoxify- 
ing action on the toxin. A sample of antipneumococcic serum con- 
centrated by the Felton method, in a 1 to 20 dilution, neutralized 
the toxin but failed to do so when added in a 1 to 50 dilution. 

The results obtained by Jamieson and Powell (1931) 678 were 
analogous to those reported by Parker and McCoy. The filtrates 
of young broth cultures of pneumococci of Types I, II, III, and 
IV, which had been found to elicit a positive reaction in the skin 
of rabbits and of human beings, constituted the antigen employed 
by subcutaneous injection for immunizing horses. The serums de- 
veloped by the procedure appeared to possess neutralizing sub- 
stances for the toxin, and these substances could be concentrated 
to a moderate degree in the refining of globulins by the usual 
salting-out methods. The concentrated serum contained only a 
small amount of protective antibody, and in neutralizing action 
compared favorably with the action of specific antitoxin on scar- 
let fever streptococcal toxin. 

Employing autolysate prepared by the method of Parker, 
Blackman 122 produced immune serum in rabbits and horses that 
would protect normal rabbits not only against the toxic action of 
autolysate but against pneumococcal infection. 

Sabin (1931) 1208 sought to determine whether antipneumotoxin 
influences the course of pneumococcal infection in mice. Mice were 
injected with large doses of Type I and II pneumococci. One 
series of animals was then treated with therapeutic antibacterial 
serum, another with antipneumotoxic serum, and a third series re- 



386 BIOLOGY OF PNEUMOCOCCUS 

ceived both antibacterial and antitoxic serums. The results showed 
that the antitoxin was without effect in staying the infection. 
Sabin then tested the action of antipneumotoxin in rabbits in- 
fected intradermally by the method of Goodner. Rabbits were 
given an intradermal injection of 0.1 cubic centimeter of a 1 to 
100 dilution of an eighteen-hour broth culture of virulent Type I 
Pneumococcus. Three groups of rabbits so injected were treated 
with the same serums as in the mouse experiments, some animals 
receiving the serum injection six or seven hours after inoculation 
and some twenty-four hours after inoculation. Of the untreated 
controls and those animals receiving antitoxin only, all died within 
the same time, and no beneficial effect was observed in the rabbits 
treated with antitoxic serum added to antibacterial serum. 

The preliminary statement of Coca (1932) 245 regarding the 
antigenic action of a pyrogenic and skin-reacting substance in 
culture filtrates of Pneumococcus in raising the resistance of chil- 
dren to the action of the antigen has already been mentioned. Not 
only did the young subjects become immune to the pyrogenic sub- 
stance and fail to react when the filtrate was injected into the 
skin, but the serum of the children so treated neutralized the ac- 
tion of the filtrate. Convalescent serum from patients recovering 
from infection with Type I and II Pneumococcus also inhibited 
the effect of the poisonous substance derived from pneumococci. 
Therapeutic antipneumococcic serum, on the contrary, in the 
amounts used, failed to neutralize minute amounts of the filtrate. 

In a more recent report (1936), Coca 246 described further ex- 
periments with filtrates of pneumococcal cultures. The toxic prin- 
ciple was neutralized by the serum of young human subjects pre- 
viously injected with the filtrates and by the serum of patients 
convalescing from lobar pneumonia. According to Coca, the neu- 
tralizing action of the human immune serum was type-specific and 
antitoxic in nature and not related to the type-specific polysac- 
charide antibody. 



ANTIBODIES TO PNEUMOCOCCUS 387 

Heterophile Antibodies 

During recent years there has developed considerable interest 
in the fact that the injection of pneumococci into suitable animals 
results in the production of heterophile antibodies. It is not neces- 
sary in this discussion to consider all the various sources and char- 
acters of heterogenetic antigens which may be used to stimulate 
the production of heterophile antibodies. Readers interested in the 
subject are referred to reviews of the subject by Davidsohn 
(1927), 294 - 7 Bull (1928), 175 and more recently and in special ref- 
erence to pneumonia, by Plummer (1936). 1099 That heterogenetic 
antigens and heterophile antibodies may be of biological signifi- 
cance in pneumococcal infections has been suggested in publica- 
tions by Bailey and Shorb (1931, 1933), 64 5 and by Powell, Jamie- 
son, Bailey, and Hyde (1933). 1105 Opposed to these views are the 
observations of Finland, Ruegsegger, and Felton (1935). 444 It is 
not proposed to relate technical details of the various experiments 
dealing with this subject. The Council on Pharmacy and Chemis- 
try of the American Medical Association requested Plummer to 
make a report on the use of heterophile antibodies and, since his 
conclusions appear sound, they are reproduced verbatim. 

The presence of heterophile bodies in animal tissues, animal serums 
and bacteria is strongly suggestive that these bodies play a part in cer- 
tain immune reactions. The animal experiments carried out by Bailey, 
Shorb, Powell, Jamieson, and Hyde do not prove that the heterophile 
bodies play a role in the pneumococcus immunity of the human being. 
Some of the results reported are open to question. The work should be 
repeated by an independent group of investigators before it is accepted. 
The study by Finland, Ruegsegger and Felton on the heterophile anti- 
bodies in the serum of patients convalescing from pneumonia and in 
controls leads one to believe that the heterophile bodies do not have any 
particular bearing on the course of pneumococcic infections in man. 
The clinical and statistical evidence cited by the manufacturers of the 
combined heterophile antibody serum is too limited and is strongly mis- 
leading. The presence of both rabbit and horse proteins in the serum 
will increase the incidence and the dangers of allergic reactions. This is 



388 BIOLOGY OF PNEUMOCOCCUS 

a definite disadvantage, because with newer methods of refining serum 
the allergic reaction is the principal source of danger in any type of 
serotherapy. There is only slight experimental evidence and no clinical 
evidence that the combined heterophile serum gives any immunity 
against Type III and Group IV pneumococcic infections. There is the 
same lack of evidence that the combined serum produces a greater im- 
munity for Type I and II infections than the ordinary antipneumococ- 
cus horse serum. The principal theoretical advantage of the combined 
heterophile serum is that it could be used for all pneumococcic pneu- 
monias, regardless of types, and the corollary of this that pneumococcus 
typing would not be necessary. Serum having this advantage would be 
highly desirable, but r because its superiority is unproved and because of 
the probable increase in allergic reactions it is unfair and unwise to 
recommend the combined heterophile serum for general distribution. 

The Council on Pharmacy and Chemistry approved and adopted 
Plummer's report and emphasized the conclusion that, in the light 
of present knowledge, recommendation of the combined heterophile 
serum for general distribution is unwise and unwarranted. 

Phagocytosis 

The part played by leucocytes in checking the invasion of pneu- 
mococci and in the destruction of cocci in the infected body was 
apparently first suggested by Gamaleia 498 in 1888. The French 
author, in a study of pneumonia patients, noted large phagocytes 
packed with cocci in various stages of degeneration. The author 
also reported an observation to the effect that sheep infected 
intratracheally with diplococci obtained from infected cadavers, 
on being sacrificed, showed many phagocytes containing the or- 
ganisms. Gamaleia ventured the conjecture that pulmonary 
phagocytes were a factor in restraining the spread of pneumococci 
{Streptococcus lanceolatus Pasteuri) in the lung. Kruse and Pan- 
sini (1891) 763 also noted phagocytosis in animals experimentally 
infected with pneumococci, but held that the phenomenon was of 
secondary importance to the bactericidal action of the serum. 

At the time, opinion was divided concerning the nature of the 



ANTIBODIES TO PNEUMOCOCCUS 389 

forces which accounted for the resistance of normal and immunized 
animals to Pneumococcus. One school contended that immunity 
was due to bactericidins ; the other maintained that the immunity 
was antitoxic in nature. The idea that mobile cellular elements — 
the leucocytes — might intervene in arresting pneumococcal infec- 
tion came as an innovation and at first was not readily accepted. 
For example, Bonome 137 shared the conception that the blood of 
immunized animals acquired increased bactericidal power for 
pneumococci and although he observed both leucocytosis and 
phagocytosis, he did not perceive that the phenomena could be 
the. basis of bactericidal action. It was Issaeff (1893) 673 who, as 
a result of his experiments, abandoned the idea that antitoxin was 
a factor, and emphasized the fact that phagocytosis played a most 
important part in acquired immunity to Pneumococcus. Mennes 
(1897) 893 was unable to demonstrate any effect of the white cells 
of normal blood on the development of pneumococcal infection. 
According to his views, the primary defensive element was the 
serum and not the leucocytes. The white cells of immune rabbits, 
however, exerted marked phagocytic action, and Mennes concluded 
that the immunity of the rabbit to pneumococcal invasion de- 
veloped from a modification of the serum, and that tiie modifica- 
tion activated the phagocytic property of the leucocytes. 

In 1904, Neufeld and Rimpau 997 definitely discarded bacteri- 
cidins and bacteriolysins as forces operating in antipneumococcic 
immunity and reported that the addition of specific immune serum 
to normal rabbit leucocytes imparted to the cells vigorous phago- 
cytic ability. The authors then extended the experiments to include 
the in vivo action of immune serum on virulent cultures injected 
into mice. When the culture alone was injected intraperitoneally, 
the organisms multiplied and only a few leucocytes were seen to 
contain cocci, but when both culture and serum were injected, a 
large number of pneumococci were engulfed by the white blood 
cells. By absorption experiments, Neufeld and Rimpau demon- 
strated that the serum acted on the cocci, whether living or killed, 



390 BIOLOGY OF PNEUMOCOCCUS 

and not on the leucocytes. Complement was not necessary to ren- 
der the organisms phagocytable. In a second report, Neufeld and 
Rimpau (1905) 998 claimed that the theory that serum was a stimu- 
lant for the leucocytes was no longer tenable, but that serum, on 
the contrary, caused a peculiar transformation of the bacteria. 
Immune serums which possessed the property of rendering bac- 
teria susceptible to phagocytosis Neufeld and Rimpau designated 
by the name "bacteriotropic" to distinguish them from bacterio- 
lytic serums. 

In America, at the time when the Wright opsonic technique was 
arousing such general interest, Rosenow 1160 tested the suscepti- 
bility of forty strains of pneumococci to phagocytosis. All but 
four strains at first resisted phagocytosis in pneumonic blood, but 
after cultivation on artificial media the organisms became suscep- 
tible to leucocytic ingestion. While heating the cocci had no effect 
on susceptibility, the opsonic property of the serum appeared to 
be diminished after a thirty-minute exposure to a temperature of 
56°. Rosenow believed that white cells from the blood of pneu- 
monia patients possessed greater phagocytic power than did nor- 
mal cells, and ascribed the pneumococcidal effect of blood to the 
combined action of serum and leucocytes — phagocytosis and in- 
traphagocytic digestion. Potter and Krumwiede (1907) 1103 tested 
the leucocytes of pneumonia patients for phagocytic properties. 
While granting the inaccuracy of the method employed, the au- 
thors, contrary to Rosenow, stated that leucocytes of patients 
during the height of pneumonic disease were probably less active 
in phagocytic power than were normal leucocytes. 

The principle in leucocytes responsible for the lysis of pneumo- 
cocci after being phagocyted was investigated by Schneider 
(1910), 1244 who believed that the hypothetical substance acted 
directly upon the injected pneumococci. Schneider also found that 
specific immune serum powerfully stimulated phagocytosis, espe- 
cially in vivo, but ascribed the action of serum to its effect on the 
white cells. Schneider further claimed that a serum which was 



ANTIBODIES TO PNEUMOCOCCUS 391 

lacking in this stimulating property was also lacking in precipi- 
tating, agglutinating, and complement-fixing action and displayed 
no protective power. Boehncke and Mouriz-Riesgo (1915), 134 who 
had previously corroborated the conclusion of Neufeld and Rim- 
pau that the bacteriotropic action of antipneumococcic serum in 
vitro also participates in the action of the serum in vivo, were not 
convinced that tropins were the only factor in inducing phagocy- 
tosis. The authors had been able to rule out any participation of 
bactericidal and complement-fixing antibodies and, while they con- 
cluded that phagocytosis was the most important factor, the au- 
thors still inclined to the view that antitoxins might play an 
important part in resistance to pneumococcal infection. In 1919, 
Barber, 77 employing the single-cell technique, sought an explana- 
tion of the ready growth of pneumococci in immune serum. Failing 
to find any specific bactericidal substance in immune serum, Bar- 
ber noticed that when specific serum was added to pneumococci 
previous to mixing with leucocytes active phagocytosis took place, 
and the same phenomenon occurred when the organisms were in- 
jected into the peritoneal cavity of mice passively immunized with 
immune serum. The author offered no explanation for the reac- 
tion. 

SENSITIZATION OF PNEUMOCOCCI 

Burgers, 188 and Burgers and Meisner (1911), 189 following the 
lead furnished by the work of Neufeld and Rimpau, found that 
while normal serum was inactive in rendering virulent pneumococci 
phagocytable, immune serum was highly active in this respect. Al- 
though the serum used in the experiments was evidently low in po- 
tency, the pneumococci when incubated with the serum were 
readily taken up by normal leucocytes. Burgers and his colleague 
believed that fresh complement was necessary for phagocytosis, 
since they considered that the reaction was one depending upon 
antigen, amboceptor, and complement. Strouse (1911 ) 1346 also 
used the method of sensitizing pneumococci in studying the pres- 



392 BIOLOGY OF PNEUMOCOCCUS 

ence of immune opsonins in the serum of pneumonia patients. The 
addition of the serum accelerated the ingestion of pneumococci by 
the white cells. 

In 1915, Friel 494 described the action of normal and immune 
serum on pneumococci. Cultures left in contact with normal serum 
for one and one-half to twenty-four hours and then washed in 
salt solution acquired no increased susceptibility to the action of 
phagocytes, but organisms similarly treated with immune serum 
were avidly ingested by the white cells. It will be recalled that the 
sensitizing effect of immune serum was called "piantication" by 
Friel. The term, meaning to prepare for slaughter, was in effect 
the same as Wright's word opsonization, meaning to prepare for 
the feast. In addition to the action of opsonins in rendering pneu- 
mococci susceptible to the destructive action of leucocytes, Good- 
ner, Dubos, and Avery (1932) 538 demonstrated that organisms 
denuded of capsule by the action of specific bacterial enzymes be- 
came highly vulnerable to phagocytosis by tissue cells. 

In an endeavor to clarify the contradictory conceptions of Neu- 
feld and Rimpau 997 on the one hand and of Romer* on the other re- 
garding the action of immune serum on bacterium or leucocyte, 
Preisz (1915), 1108 by employing normal and immune serum and 
leucocytes in both in vitro and in vivo experiments, concluded that 
the ability of immune serum to promote phagocytosis of pneumo- 
cocci lay in its action on the cocci and not on the leucocytes. 

A more recent report — that of Robertson and Sia (1927) 1147 — 
dealt with the action of the serum of both naturally resistant and 
susceptible animals on the phagocytability of Pneumococcus. 
When virulent cultures were sensitized by contact for one hour at 
37° with the serum of animals resistant to Pneumococcus, the or- 
ganisms were actively ingested not only by homologous leucocytes 
but also by the white blood corpuscles of other resistant and sus- 
ceptible animals. On the contrary, when pneumococci were exposed 
to the action of the serum of animals normally susceptible to pneu- 

* Quoted by Neufeld and Haendel.992 



ANTIBODIES TO PNEUMOCOCCUS 393 

mococcal infection, the cocci were not taken up by the leucocytes 
of either resistant or susceptible animals. The serum of all the re- 
sistant species tested — dog, cat, sheep, pig, and horse — possessed 
marked opsonic properties not found in the serum of animals of 
susceptible species, such as the rabbit, guinea pig, and man. There 
appeared, however, to be no essential difference in the phagocytic 
activity of the leucocytes from the various animals. Heating the 
serum, according to Robertson and Sia, abolished the destructive 
power of serum-leucocyte mixtures for pneumococci. The validity 
of the authors' classification of resistant and susceptible animals 
is open to question. To group the horse with such highly resistant 
animals as the dog, cat, sheep, and pig, and to include species of 
such diverse susceptibility in the non-resistant class, may not be 
wholly justified. In another portion of the report, Robertson and 
Sia drew attention to other conditions which may influence the 
opsonizing property of serum on pneumococci subjected to the ac- 
tion of leucocytes. The strain and type of the culture employed, 
the age of the animals, and other factors discussed in the chapter 
on Pathogenicity must be taken into account in evaluating the 
results. 

From the conclusions in an extensive report on an investigation 
of experimental pneumococcal septicemia and antipneumococcal 
immunity published by Wright in 1927 , 1647 certain passages may 
be cited. Avirulent pneumococci of Type I inoculated intrave- 
nously into rabbits are rapidly removed and do not reappear in 
the blood; virulent strains of the same serological type similarly 
inoculated disappear for a short period and then subsequently in- 
crease in number. Previous active immunization enhances the ca- 
pacity of the rabbit to remove virulent organisms and to prevent 
their reappearance, and in the immunity so established the out- 
standing effect is the increased activity of the body fluids favor- 
ing phagocytosis, although the existence of a slight degree of 
residual and purely cellular immunity cannot be excluded. Because 
leucocytes could be considerably decreased in number without in- 



394 BIOLOGY OF PNEUMOCOCCUS 

terfering with the capacity of the animal to dispose of organisms 
introduced into the blood, taken in connection with the specific ac- 
tion of serum on the cocci, Wright laid particular stress on the 
importance of humoral immune elements in bringing about the 
destruction of invading pneumococci. 

That circulating antibodies and not leucocytes play the domi- 
nant part in recovery from pneumococcal infection was also the 
contention of Robertson, Woo, Cheer, and King (1928). 1152 The 
blood of cats and rabbits surviving experimental pneumococcal in- 
fection possessed the ability to promote the destruction of highly 
virulent pneumococci in rabbit serum-leucocyte mixtures, which 
mixtures in themselves had no growth-inhibitory action. The bac- 
tericidal action of the serum was associated with a marked in- 
crease in acquired resistance to infection. In cats, which were 
studied in greater detail, the pneumococcidal promoting power of 
the serum as well as the opsonic, agglutinative, and mouse-pro- 
tective properties, which were found to be type-specific, became 
demonstrable at the time of recovery, and their appearance in the 
serum always marked the termination of blood invasion. The ani- 
mals succumbing to infection failed to develop detectable immune 
properties in the serum and showed persistent bacteriemia. Ac- 
cording to the authors, the degree of leucocytosis had no constant 
relation to the outcome of the disease. 

In two communications, Terrell ( 1930 ) 1385 " 6 presented the results 
of experiments carried out to determine the changes in humoral 
immunity occurring during the early stages of experimental pneu- 
mococcal infection. Using the technique of Robertson and Sia 1144 " 8 
to determine circulating antibodies, the author, after infecting 
normal cats and dogs with virulent cultures of Type I and II 
pneumococci, found that in a generalized and overwhelming infec- 
tion accompanied by early blood invasion, there was a prompt and 
rapid decrease in the concentration of native humoral immune 
bodies, which frequently disappeared entirely by the time of death. 
Animals surviving a moderately severe, generalized infection 



ANTIBODIES TO PNEUMOCOCCUS 395 

showed a similar early diminution of humoral immune substances, 
but with the onset of recovery the concentration of the immune 
bodies again rose. When the infection was localized, as in the case 
of true lobar pneumonia, the presence of humoral antibodies in 
quantity could be demonstrated in the blood throughout the course 
of an infection terminating in death. 

Other evidence indicating the participation of leucocytosis in 
resistance to pneumococcal infections is to be found in the com- 
munication of Schattenberg and Harris (1932). 1222 The plan of 
the experiments was to induce leucocytosis in white mice by pre- 
liminary injections of killed cultures of typhoid bacilli or staphy- 
lococci, of detoxified suspensions of devitalized pneumococci, and 
of sterile milk. Six to eight hours later the animals were inocu- 
lated intraperitoneally with lethal doses of pneumococci of Types 
I, II, and III so measured that death of the mice could be con- 
trolled to take place at periods varying from three to twenty-four 
hours. The animals thus treated failed to show that stimulation of 
leucocytosis had any effect in preventing the development of pneu- 
mococcal peritonitis. 

NORMAL TROPINS OR OPSONINS 

The ability so to sensitize pneumococci that they become attrac- 
tive to leucocytes and susceptible to phagocytic lysis is a charac- 
ter of some normal as well as of immune serums. Rosenow 1160 " 1 dis- 
closed the property in normal human serum and found opsonins 
for streptococci, staphylococci, and tubercle bacilli, in addition to 
those for pneumococci. 

Ungermann (1911) 1434 was unable to discover any phagocytosis 
of virulent pneumococci in the serum of normal mice and rabbits. 
Avirulent strains were readily phagocyted, and the degree of the 
action appeared to parallel the resistance of the particular species 
of animal whose serum was used. The conclusion was reached that 
normal resistance depended upon the phagocytic power of fresh 
normal serum, at least in the cases studied, and that the serum must 



396 BIOLOGY OF PNEUMOCOCCUS 

develop this power in vitro in the presence of leucocytes. Woo 1541 
later (1926) found, in testing the pneumococcidal activity of nor- 
mal serum-leucocyte mixtures, that virulence of the cultures and 
the age of the test rabbit influenced the outcome of the test. 
Avirulent strains were readily destroyed in a mixture of normal 
rabbit serum and leucocytes, whereas virulent cultures resisted the 
destructive action of the mixture. The absence of pneumococcidal 
properties in the blood of very young rabbits agreed with the ex- 
treme susceptibility to pneumococcal infection of immature ani- 
mals of the species. 

Strouse, 1346 by both in vitro and in vivo tests, demonstrated the 
same quality in the peritoneal fluid of pigeons. Wright 1547 noted 
the rapid disappearance of virulent pneumococci injected directly 
into the blood stream of rabbits and the still more rapid removal 
of avirulent forms. Specific immunization enhanced the activity of 
body fluids favoring phagocytosis. Robertson and Sia, 1144 and 
later Sia, 1268 not only proved the existence of normal opsonins for 
pneumococci in the blood of cats, dogs, sheep, and pigs but, by ab- 
sorption experiments, exhausted the serum of opsoninizing sub- 
stance for the strain used as absorbent without removing opsonins 
for organisms of other types. The results suggest that there are 
separate type-specific opsonins for pneumococci in the serum of 
animals naturally resistant to pneumococcal infection. 

In 1933, Ward and Enders 1484 published a communication, based 
on experiments with Pneumococcus, dealing with a serological 
analysis of the opsonic or tropic action of normal and immune se- 
rum. The authors studied the action of the anticarbohydrate sub- 
stance in normal serum as an opsonic or tropic agent, and also the 
properties of normal and immune serum which promote phago- 
cytosis after the anticarbohydrate substance or antibody had been 
removed from the serum by appropriate amounts of type-specific 
polysaccharide. The authors analyzed the mechanism of the 
phagocytosis of Pneumococcus as follows: In normal, unheated 
human serum virulent pneumococci may be prepared for phago- 



ANTIBODIES TO PNEUMOCOCCUS 397 

cytosis by two separate antibodies acting in conjunction with 
complement. One of these is the type-specific anticarbohydrate 
antibody reacting with the carbohydrate fraction of Pneumococ- 
cus ; the other is probably also a type-specific antibody, but quite 
distinct from the former and, therefore, reacting with a different 
antigenic constituent of the bacterium. In normal human serum 
heated to 56° these two antibodies may, after prolonged contact 
with the organism, promote phagocytosis of Pneumococcus with- 
out the adjuvant action of complement. The two antibodies are 
equally effective in the phagocytosis of twenty-four-hour cultures 
by normal blood, but the anticarbohydrate antibody tends to pre- 
dominate as the pneumococci approach the state in which they 
exist in the animal body. The anticarbohydrate antibody was the 
only one in immune serum which could be demonstrated to induce 
phagocytosis. It was active by itself, but complement enhanced its 
effect. 

To Ward and Enders it seemed that a single well-defined anti- 
body — the anticarbohydrate antibody — might be responsible for 
the phagocytic action of unheated normal serum, of heated normal 
serum, inactivated immune serum, and immune serum activated by 
complement. The facts appeared to invalidate the division pro- 
posed by Neufeld of the phagocytic antibodies into bacteriotro- 
pins — antibodies, the phagocytic titer of which is not raised by 
the addition of complement — and opsonic antibodies — antibodies, 
comparable to lysins, which are only active in the presence of 
complement. Complement alone was found to be incapable of in- 
ducing phagocytosis of Pneumococcus and, therefore, may act 
merely as a catalyst in increasing the velocity of the phagocytic 
process. On the basis of their observations, Ward and Enders pro- 
posed that the term "tropin" be discarded, since it was misleading 
and unnecessary, and that the term "opsonin" be retained to de- 
note any heat-stable antibody which prepares bacteria for phago- 
cytosis. Contrary to current usage the latter term would not sug- 
gest a combination of antibody with complement. 



398 BIOLOGY OF PNEUMOCOCCUS 

ANTIOPSONINS 

The existence of substances in Pneumococcus inimical to leuco- 
cytic ingestion became apparent to Rosenow (1907) 1161 who, by 
saline extraction or by autolysis of the cocci, obtained a substance 
or substances that inhibited opsonic action. It was found further 
that avirulent strains could absorb the inhibiting substance and 
then become resistant to phagocytosis, while after similar extrac- 
tion virulent organisms acquired the capacity to absorb opsonin 
and became vulnerable to the destructive action of the white blood 
cells. The presence of substances in pneumococci capable of inhib- 
iting phagocytosis was demonstrated by Tchistovitch and Youre- 
vitch 1383 in 1908. When saline suspensions of washed, virulent 
pneumococci were added to serum-leucocyte mixtures, marked 
phagocytosis resulted, but when a small amount of the washings 
from the original culture was added to the combination the cocci 
were no longer ingested by the leucocytes. The inhibiting sub- 
stances were found only in cultures of virulent strains, they were 
specific for the bacterial species and were thermostable. For such 
substances Tchistovitch proposed the name "Antiphagins." 

Pritchett 1110 endeavored to develop antiopsonins in rabbits by 
injecting them with antipneumococcic horse serum, but could ob- 
tain no evidence of the formation of substances antagonistic to the 
action of immune serum. On the contrary, the serum of rabbits in- 
jected with antipneumococcic horse serum for Type I, II, or III 
pneumococci, when combined with antipneumococcic serum for 
Types I and II, increased opsonization of organisms of Types I 
and II but in this respect never affected strains of Type III. 

Confirmation of the existence of antiopsonic principles in viru- 
lent pneumococci is to be found in the experiments of Wadsworth 
and Sickles, 1475 and of Sickles (1927). 1277 Intracellular substances 
released by sodium oleate solution of the organisms, or present in 
the sterile filtrates of broth cultures, prevented phagocytosis of 
pneumococci in immune-serum and leucocyte mixtures. There was 
some evidence of type-specificity in the action of the pneumococcal 



ANTIBODIES TO PNEUMOCOCCUS 399 

derivatives, since an extract of Type I Pneumococcus failed to 
prevent phagocytosis of Type III organisms in the presence of 
Type III antiserum, and vice versa. The inhibiting substance af- 
fected the organisms and not the leucocytes. Furthermore, the ac- 
tive principle could be absorbed from extracts by combination with 
specific immune serum. 

Yamamoto (1929) 1557 " 8 studied the effect of both unheated and 
heated culture filtrates of pneumococci on spontaneous phagocy- 
tosis in vivo in the rabbit. The "Impedin," as the author called the 
inhibitory principle, was without action on the number of mi- 
grated leucocytes. The injection of heated filtrates affected phago- 
cytosis in inverse ratio to the duration of previous exposure of the 
filtrate to heat, the antiopsonic property being completely de- 
stroyed after thirty to sixty minutes at boiling temperature. 

The action of the specific capsular polysaccharide, so conspicu- 
ous in the inhibition of other serological reactions between Pneu- 
mococcus and immune serum, is manifested in its antagonistic ef- 
fect on opsonins, and is referable to its function of combining with 
and precipitating the antibodies from serum. In studies on growth 
inhibition, Sia (1926), 1267 by means of normal rabbit or cat se- 
rum-leucocyte mixtures, learnt that the presence of a very small 
amount of purified soluble specific substance from pneumococci of 
both Types I and II markedly altered the conditions in the mix- 
ture so that even a small number of avirulent pneumococci were 
enabled to grow in the presence of serum and leucocytes of animals 
which ordinarily possess the power to destroy the organisms in 
relatively large numbers. The action of the capsular carbohydrate 
was type-specific, and the same neutralizing effect on the growth- 
inhibitory or pneumococcidal power of normal serum-leucocyte 
mixtures was exhibited by broth filtrates of cultures of young 
pneumococci. 

Ward 1480 " 1 confirmed Sia's observations on the specific anti- 
phagocytic action on blood of soluble specific substance, and found 
the action more marked in the case of Type III than Type I pneu- 



400 BIOLOGY OF PNEUMOCOCCUS 

mococci. In in vitro phagocytic experiments with human blood, 
antipneumococcic serum, capsular polysaccharide, and living, 
virulent pneumococci, there was a zone of definite phagocytic 
inhibition when a strong antiserum was used. Upon dilution of the 
serum there appeared a zone in which phagocytosis was effective. 
Ward suggested that the inhibition was caused by the specific pre- 
cipitate formed by the combination of soluble specific substance 
and precipitin, which interfered, perhaps mechanically, with the 
ingestion of the pneumococci by leucocytes. 

In later reports, Ward 1482 " 3 described a type-specific substance 
with a powerful antibacterial action present in the filtrate of a 
five-day broth culture of Type III Pneumococcus. A similar sub- 
stance was also demonstrated in the filtrate of a lung obtained at 
necropsy from a patient dying of lobar pneumonia caused by a 
Type III organism. Ward employed a method devised by himself 
for estimating the pneumococcidal action of whole blood, and 
found that if the precipitinogen content of the broth filtrate and 
the amount of soluble specific carbohydrate of Type III Pneumo- 
coccus were taken as a basis of comparison, it required approxi- 
mately one thousand times as much antiserum to neutralize the 
antagonistic action of the broth filtrate as was necessary to neu- 
tralize the specific carbohydrate. Ward also found that a specimen 
of blood from a patient convalescing from Type III lobar pneu- 
monia, though comparatively weak in anticarbohydrate antibody 
(precipitin), was better able to neutralize the broth filtrate and 
the lung filtrate than a corresponding mixture of normal blood and 
specific antipneumococcic serum. 

Enders and Wu (1934) 362 more recently reported that the op- 
sonic titer of normal serum could be practically abolished by the 
addition of the A carbohydrate. In immune serum, the A sub- 
stance brought about a quantitatively greater reduction in op- 
sonic activity than did its derivatives, but the authors were never 
successful in demonstrating complete inhibition of phagocytic ac- 
tion by the method of absorption of antibody. Enders and Wu also 



ANTIBODIES TO PNEUMOCOCCUS 401 

showed that the A carbohydrate was far more effective as an anti- 
bactericidal — that is, antiopsonic — agent than deacetylated de- 
rivatives of the capsular polysaccharide. 

OPSONINS IN PNEUMONIA 

Among others, Rosenow (1906) 1160 announced that the content 
of opsonins for Pneumococcus appeared to be less in the serum of 
patients succumbing to pneumonia than it was in normal human 
serum or in serum of patients during or after crisis. Using the 
Neufeld technique, Strouse (1911) 1346 could demonstrate no phago- 
cytosis of virulent pneumococci by serum obtained from pneu- 
monia patients after crisis, but by sensitizing the organisms with 
convalescent serum previous to the test, phagocytosis was ob- 
served in about one-fourth of the cases. Eggers (1912), 349 employ- 
ing the plate method of cultivation, noted an increase in the pneu- 
mococcidal power of the blood of pneumonia patients at or near 
the time of crisis, and while he demonstrated that the action was 
due to phagocytosis, Eggers presented no detailed experimental 
evidence to prove whether the action of serum was exerted on the 
cocci or on the leucocytes. Clough (1913) 240 observed that the se- 
rum of approximately one-half the pneumonia patients obtained 
after crisis or lysis exhibited definite phagocytic activity, and that 
the reaction was strictly limited, under the conditions of the test, 
to the homologous strain of Pneumococcus isolated from the pa- 
tient whose serum was being examined. Clough concluded that, 
since the active substances resisted heating at 56° and persisted in 
the serum in vitro for a considerable period of time, the substances 
must be tropins and, since phagocytic activity of the serum ap- 
peared to parallel closely the protective power for mice in inci- 
dence, time of appearance, and strain specificity, the protective 
action of serum was directly due to its ability to promote phago- 
cytosis. In a second report, Clough (1919) 242 confirmed his earlier 
observations and added that while in 85 per cent of cases of acute 
lobar pneumonia the serum showed positive phagocytic activity 



402 BIOLOGY OF PNEUMOCOCCUS 

after crisis or lysis, it lacked the property in the great majority 
of instances where the patient had died of the disease. The height 
of bacteriotropic activity of the serum of pneumonia patients was 
found by Adler (1923) 3 to occur at the time of crisis. 

THE MECHANISM OF PHAGOCYTOSIS 

The factors and processes involved in the response of rabbits to 
intravascular injection of pneumococci were investigated by Kita- 
gawa (1915). 717 When the living organisms were injected directly 
into the blood stream of rabbits there was an immediate, initial 
drop in the count of circulating cocci, followed by either a slow 
increase or decrease in number depending upon the dosage and viru- 
lence of the injected culture. When similar injections were made 
into actively immunized rabbits, the cocci disappeared with great 
rapidity from the circulation, the blood usually becoming sterile 
within ten minutes after the injection. Kitagawa concluded that 
the speedy disappearance of the injected pneumococci from the 
blood stream of actively immunized rabbits was not due to destruc- 
tion of the organisms by plasma or leucocytes, but rather to their 
mechanical removal or destruction by the fixed tissues. Kline and 
Meltzer (1915) 727 also noted that pneumococci promptly vanished 
from the pneumonic lung of dogs infected by intrabronchial in- 
sufflation after previous repeated intravenous injections of pneu- 
mococci. 

The same phenomenon was studied by Winternitz and Kline 
(1915). 1522 In normal rabbits the reaction to inoculation was im- 
mediate and the ultimate result was bacteriemia and death ; in pas- 
sively immunized animals the organisms rapidly disappeared from 
the blood and recovery took place; in rabbits previously rendered 
aplastic with benzol and then injected with specific immune serum, 
there was an immediate effect on the injected pneumococci, but 
bacteriemia recurred and death ensued; in actively immunized, 
aplastic rabbits, the initial reaction was the same as in the pas- 
sively immunized, aplastic rabbits, but the actively immunized, 



ANTIBODIES TO PNEUMOCOCCUS 403 

aplastic rabbits recovered. Winternitz and Kline accordingly 
concluded that the immunological response was dependent upon at 
least three factors, namely, immune bodies, white blood cells, and a 
third factor relying for its existence on the presence of white 
blood cells at the time of inoculation with Pneumococcus. The hu- 
moral immune bodies acted in causing the immediate disappear- 
ance of the injected organisms from the circulation, but the third 
factor, originating in the leucocytes, appeared to be essential to 
the recovery of the infected animal. 

The importance of elements peculiar to immune serum for the 
protection of mice was shown by Tilgren (1915) 14012 who, by add- 
ing immune sheep serum and sheep leucocytes to an infective dose 
of pneumococci, protected rabbits against infection. In mice, the 
immune serum afforded protection but the addition of heterolo- 
gous leucocytes resulted in no increase in the protective power of 
the serum. 

More detailed information regarding the tissues concerned in re- 
sistance to pneumococcal infections is to be found in the communi- 
cation of Permar (1923), 1082 who believed that the mononuclear 
phagocytic cells appearing in the exudate in acute experimental 
pneumonia in the rabbit were of vascular endothelial origin. Other 
cells of visceral origin possibly contributed in some degree to the 
total number of phagocytes in the exudate. Dust cells of similar 
origin already present in the alveoli also might act as phagocytes 
in inflammatory reactions, while interstitial dust cells could be- 
come reactivated and through liberation by inflammatory edema 
were enabled to appear in the exudate. The entire group of cells, 
therefore, including the newly produced phagocytes and the dust 
cells, may take part in the reaction to the invading pneumococci. 

Singer and Adler (1924), 1290 after studying the response in rab- 
bits to infection with Type III Pneumococcus, believed that the 
defensive factors resided in the reticuloendothelial system, and 
that the histocytes of bone marrow, the capillary endothelia of the 
parenchymatous organs, the endothelium of serous cavities, and 



404 BIOLOGY OF PNEUMOCOCCUS 

the biologically comparable alveolar epithelia, as well as the fixed 
tissue cells with phagocytic properties underlying them, must have 
experienced in the immune animal a specific alteration in the sense 
of acquiring the power to ingest pneumococci and to retain and 
eventually to kill the organisms. To Singer and Adler, leucocytic 
phagocytosis merely exerted a secondary chemotactic action, but 
had nothing to do with specific immunity toward a given organ- 
ism. In a second communication in the same year, Singer and Ad- 
ler 1291 reported analogous results obtained in experiments of the 
same nature with pneumococci of Types I and II. The authors 
maintained that in passive immunity the cellular system played the 
main part, the action of the serum being purely a bacteriotropic 
one. The phenomenon was definitely type-specific and the elements 
of the reaction were located in the reticuloendothelial tissues, 
since functional blockade of that system caused a disappearance 
of the manifestations of immunity. 

Tudoranu 1427 disagreed (1926) with the theory of Singer and 
Adler that immunity toward Type III Pneumococcus was devoid 
of specific humoral elements and quoted the latter's own experi- 
ments to refute the claim. Although in passively immunized rab- 
bits injected with Type III cultures the organisms for a time mul- 
tiplied in the blood, the animals eventually recovered, whereas 
normal rabbits similarly inoculated succumbed to infection. Tu- 
doranu, in support of the contention, cited his success in passively 
immunizing rabbits by the subcutaneous, intraperitoneal, and in- 
travenous injection of specific immune serum, and suggested that 
the successful result was merely a question of dosage of serum. 
The author concluded that the action of the serum was to neutral- 
ize the aggressins, thus permitting the production of an exudate 
rich in leucocytes, which modified the cocci in such a way as to 
render phagocytosis possible. 

In the interval between the publication of the papers of Singer 
and Adler and of Tudoranu, Neufeld and Meyer (1924) 995 re- 
ported the results of a study of the origin of antipneumococcal 



ANTIBODIES TO PNEUMOCOCCUS 405 

immunity. According to the latter authors, actively immunized 
mice manifested the same specific phagocytosis as those passively 
immunized, but scarcely ever had demonstrable protective bodies 
in the blood. However, after intravenous injection of manganese 
salts, the protective substances appeared in the blood in large 
amounts. Neufeld and Meyer concluded that the active immunity 
depended entirely upon antibodies, that the antibodies were formed 
in the reticulo-endothelium and that, in a broader sense, the cells 
of the Gefassbindegewebsapparat were the sole source of anti- 
bodies. 

The ability of macrophages to dispose of pathogenic bacteria 
was described by Nakahara (1925). 941 The method of demonstrat- 
ing the action of these cells was the injection of olive oil into the 
peritoneal cavity of mice. The injection was followed at first by 
the appearance of polymorphonuclear leucocytes, which were 
largely replaced by mononuclear cells. When pneumococci were in- 
jected into mice during the macrophagic reaction, the organisms 
were destroyed more rapidly than was the case in normal animals, 
and the mice survived multiples of the minimal infecting dose. The 
macrophages were observed to phagocyte the injected cocci ac- 
tively. 

Meyer (1926), 896 after blocking the reticuloendothelial system 
by the intravenous injection of ferric saccharate, India ink, trypan 
blue, and other dyes, along with the removal of the spleen, was 
unable, as a rule, to immunize mice by the injection of killed pneu- 
mococci of Type I. When active immunization preceded the re- 
moval of the spleen and the injection of the blocking agents, the 
immune condition of the animals was sometimes altered. Meyer 
was not able to substantiate the earlier observation of Neufeld 
and Meyer on the necessity of stimulating the endothelia by man- 
ganese, since protective antibodies were found to be present in the 
blood of the majority of mice immunized with Type I pneumococci. 
Inasmuch as splenectomized mice in which the reticulo-endothelial 
system had been blocked easily acquired passive protection after 



406 BIOLOGY OF PNEUMOCOCCUS 

the injection of specific immune serum, Meyer argued that the ef- 
fect of these measures could not be due to a decrease in the phago- 
cytic activity of the reticuloendothelial cells, but rather to an in- 
hibition of antibody formation in the cells and that active as well 
as passive immunity depended upon specific antibodies circulating 
in the blood and present in the cells. The contribution of the re- 
ticulo-endothelial system to the operation of the immune mecha- 
nism was believed by Wright (1927) 1547 to be of relatively slight 
importance. Although he acknowledged that the possibility of a 
residual cellular immunity could not be overlooked, Wright con- 
sidered circulating antibodies to be the prime factor in immunity 
to Pneumococcus. 

The relationship of blood-free splenic cells of normal and im- 
munized rabbits was investigated by Loewenthal and Micseh 
(1929). 822 Using the technique of tissue culture devised by the 
first-named author, it was found that the macrophages of the 
spleen of normal rabbits phagocyted avirulent pneumococci only 
in the presence of fresh, normal serum, and phagocyted virulent 
organisms only in the presence of specific immune serum. Macro- 
phages of the spleen of immunized rabbits phagocyted both aviru- 
lent and virulent pneumococci without the addition of either nor- 
mal or immune serum, when the tissue fragments were not trans- 
planted into a fresh medium. However, when bits of spleen tissue of 
immunized rabbits were transplanted into fresh media during tis- 
sue culture, the macrophages behaved similarly to those of normal 
animals. Loewenthal and Micseh believed that the results pointed 
to a close relation between the mechanisms of humoral and cellular 
immunity. 

In 1932 and 1933, Kritschewski, Rubinstein, and Heronimus 758 ' 9 
confirmed the results of Meyer on the inability of antipneumococ- 
cic serum to protect mice that had been splenectomized and had 
suffered artificial impairment of function of the reticuloendo- 
thelial system. The great majority of mice so treated succumbed 
to intraperitoneal inoculation with Type I Pneumococcus. 



ANTIBODIES TO PNEUMOCOCCUS 407 

Protective Antibodies 

To resolve the protective action of antipneumococcic serum into 
the causal elements participating in the phenomenon is a difficult 
task. There is the innate defense mechanism of certain animal spe- 
cies ; the functional ability of various animals to respond to anti- 
genic stimuli, as manifested by the elaboration of antibodies of dif- 
ferent properties ; the interaction of tropins or opsonins, cocci, 
and white blood corpuscles ; and the natural or the artificially en- 
hanced avidity of fixed tissue cells for pneumococci. As in other 
aspects of antipneumococcal immunity, the communications of the 
Klemperers (1891) 724 " 5 were prophetic in suggesting that the se- 
rum of animals after being injected with derivatives of Pneumo- 
coccus developed the ability to render the cocci susceptible to the 
action of blood cells and body cells and, therefore, harmless to the 
injected animal. The authors erroneously ascribed the protective 
action of the serum to an antitoxic effect, but the assumption does 
not invalidate the observation. Eyre and Washbourn (1898), 374 by 
injecting mice with mixtures of varying amounts of antipneumo- 
coccic horse serum and virulent cultures of pneumococci, were the 
first workers to demonstrate quantitatively the protective power 
of specific immune serum. The authors later applied the method to 
the standardization of therapeutic serum and, in doing so, discov- 
ered that the method could be employed to distinguish between dif- 
ferent varieties of the organism.* 

Neufeld and Haendel (1910), 991 using the protection test for 
evaluating the strength of immune serum, confirmed the observa- 
tions of Eyre and Washbourn on the existence of types of pneu- 
mococci with different serological affinities and thus established the 
protection test as a means for the classification of pneumococci 
into different serological types. Neufeld and Haendel at first em- 
ployed 0.2 cubic centimeter of immune serum, which they in- 

* Wassermann (1899)i*9i sought to locate the origin of protective substances, 
and from his experiments concluded that the antibodies apparently were 
formed in the bone marrow, and that the lymph nodes, thymus, and spleen 
merely served as reservoirs. 



408 BIOLOGY OF PNEUMOCOCCUS 

jected intraperitoneally into mice weighing 18 to 20 grams two 
to three hours before giving an intraperitoneal inoculation of 
varying amounts of virulent broth cultures. Later, when more po- 
tent serum became available, falling amounts of serum were in- 
jected into mice just previous to inoculation with one or more 
multiples of the minimal infecting dose. Ungermann and Kan- 
diba 1438 applied the Neufeld and Haendel technique to a study of 
the quantitative relations between the protective strength of im- 
mune serum and the amount of culture used, and discovered that 
the action of the serum failed to follow the law of multiple propor- 
tions. When the tests were performed on rabbits, there appeared to 
be a definite relation between the volume of serum and the weight 
of the animal, called by Ungermann and Kandiba (1912) the 
Schwellenwert or threshold value. Above this value serum pro- 
tected against many multiples of the minimal lethal dose of cul- 
ture, but below the threshold the serum had little if any action. In 
their experience, Neufeld and Haendel (1912) 993 found that, in 
order to ensure protection with serum, it was necessary to cross 
the hypothetical threshold and to administer large volumes of 
serum. 

SPECIFICITY 

Some claims have been advanced concerning the lack of type- 
specificity in the protective mechanism. For example, Yoshioka 
(1923) 1561 reported that mice that had survived an intraperitoneal 
inoculation of pneumococci were protected against an intraperi- 
toneal injection three or four days later of cultures of heterolo- 
gous type. Furthermore, mice similarly treated were able to with- 
stand inoculation with streptococci, while large doses of killed 
staphylococci produced definite protection against pneumococci. 
Yoshioka also noted a certain degree of cross-protection in guinea 
pigs and rabbits after pneumococcal infection. Kolchin and Gross 
(1924*) 739 observed cross-protection for Type III strains in a sam- 
ple of Felton's monovalent Type I antipneumococcic serum. Mono- 



ANTIBODIES TO PNEUMOCOCCUS 409 

valent rabbit antiserum prepared with pneumococci of Types II 
and III also exhibited cross-protection between Type III serum 
and Type I organisms. Despite the heterologous action observed 
in the protection test, all the samples of immune rabbit serum 
tested were found to be type-specific in agglutinin content. 

From the data presented in the communications of Yoshioka 
and of Kolchin and Gross it is difficult properly to interpret the 
results. However, against the claims of the occurrence of cross- 
protection with monovalent immune serum there is a mass of evi- 
dence attesting the specificity of the reaction when serum is pre- 
pared from carefully authenticated strains of Pneumococcus and 
when similarly identified strains are used for testing the protective 
action of immune serum. The mouse protection test, when prop- 
erly executed, therefore, remains the final criterion for establish- 
ing the type-identity of pneumococcal strains. 

RELATION TO OTHER ANTIBODIES 

A definite relation exists between the amount of protective anti- 
body in a given serum and its content of agglutinins and precipi- 
tins. Among the many references which might be cited, the work of 
Heidelberger, Sia, and Kendall (1930) 630 and the study of Felton 
(1931 ) 407 are chosen as illustrative. The former authors deter- 
mined a close parallelism between the specifically precipitable pro- 
tein and the number of mouse-protective units in a wide variety of 
Type I antipneumococcic serums. So constant were the results 
yielded by the simple method for determining the amount of pre- 
cipitable protein in immune serum that the reaction as described 
in the section of the present chapter dealing with precipitins could 
be relied upon to measure the protective strength of the serum. 
Sabin (1931) 1204 was unable to precipitate the totality of protec- 
tive antibody from antipneumococcic serum by the addition of 
homologous soluble specific substance and hence assumed the exist- 
ence of a protective substance distinct from the anticarbohydrate 
antibody. However, Felton 407 found in samples of Type I anti- 



410 BIOLOGY OF PNEUMOCOCCUS 

pneumococcic horse serum a close correlation between protection, 
precipitin, and agglutination titer for Type I Pneumococcus.* 
Similarly, Barnes, Clarke, and Wight (1936), 82 using a modified 
precipitin test, found that the correlation between precipitin and 
protective antibody content was sufficiently close to warrant the 
substitution of the precipitin test for the animal test in titration 
of the protective value of antipneumococcic serum.* 

ADDITIONAL DATA 

Kyes (1911) 765 employed domestic fowls for the preparation of 
antipneumococcic serum, giving the birds repeated inoculations of 
large doses of virulent pneumococci. Kyes concluded that by this 
method it was possible to obtain serum possessing distinct protec- 
tive action against pneumococci within certain hosts. In 1920, 
Wadsworth, 1460 after injecting a single dose of standardized pneu- 
mococcal vaccines of Types I, II, and III into mice, found that it 
was possible with Type I vaccine to produce protection against 
Type I cultures but not against Type II or III strains. In the case 
of Type II vaccine there appeared to be protection exclusively 
against the homologous organism while, when Type III vaccine 
was administered, there was no protection against any of the three 
types. Cecil and Blake, 206 after injecting monkeys with saline sus- 
pensions of heat-killed virulent Type I pneumococci, could demon- 
strate protective antibodies in the serum although the animals 
eventually succumbed to intratracheal inoculation with the same 
living strains. Later, Cecil with Steffen, 213 by the intravenous and 
subcutaneous injection of larger doses of pneumococcal vaccine, 
succeeded in completely protecting monkeys against infection and 
made the seemingly paradoxical observation that protective sub- 
stances might or might not be present in the serum of the immune 
monkeys. 

The same authors 214 reported (1925) a similar condition in 
monkeys immunized by intratracheal vaccination, and decided that 

* See page 378 of this chapter. 



ANTIBODIES TO PNEUMOCOCCUS 411 

the immunity so produced was probably cellular in nature. Ba- 
rach, 74 employing mice and rabbits, was able by the injection of 
heat-killed pneumococci of Types I and II, and of sterile culture 
filtrates of the organisms, to demonstrate the presence of protec- 
tive antibodies in the serum of the animals three days after injec- 
tion. The degree of immunity produced was greater in the case of 
Type I than of Type II vaccine, and Barach concluded that im- 
munity was due to the elaboration of protective antibodies. Ba- 
rach (1931) 76 obtained analogous results after injecting similar 
vaccines prepared from heterologous strains of pneumococci into 
patients suffering from lobar pneumonia. The average time of ap- 
pearance of protective antibodies was between five and six days 
after intravenous and intradermal administration of the killed 
pneumococci. Similar effects followed the intradermal injection of 
protein-free, type-specific polysaccharide from pneumococci of 
Types I, II, and III into normal individuals, as reported by Fin- 
land and Sutliff (1932). 448 

In the year previous, Avery and Goebel 45 had demonstrated that 
the appearance of type-specific protective antibodies followed the 
injection into rabbits of the capsular polysaccharide of Type III 
Pneumococcus conjugated with horse-serum globulin, and that it 
was the polysaccharide which determined the type-specificity of 
the ensuing protective antibodies. Avery and Goebel 46 then (1933) 
proved that the acetyl polysaccharide could induce the develop- 
ment of the specific protective substance in animals. Confirmation 
of the formation of type-specific protective antibodies after injec- 
tion of the capsular polysaccharide was forthcoming from the 
study of Francis (1934) 474 on the immunizing effect of the intra- 
dermal injection of small amounts of pneumococcal carbohydrate 
into man. 

CHEMICAL NATURE OF PROTECTIVE ANTIBODIES 

The work of Avery (1915) 32 yielded the first information con- 
cerning the particular protein element in antipneumococcic serum 



412 BIOLOGY OF PNEUMOCOCCUS 

with which the specific protective antibodies are associated, and 
furnished the foundation for the future development of methods 
for the isolation and concentration of the protective substance. 
From the serum of horses immunized with pneumococci of Types I 
and II over a period of one to two years, Avery was able, by add- 
ing ammonium sulfate to 38 to 42 per cent saturation, to precipi- 
tate completely the immune bodies. The protein fraction holding 
the protective antibodies did not correspond to the euglobulin of 
the serum since the fraction was not rendered insoluble by one- 
third saturation with ammonium sulfate or by complete saturation 
with sodium chloride. In 1924, Felton, 896 by the use of water acidu- 
lated with tartaric acid, separated the bulk of protective sub- 
stance from antipneumococcic serum of Types I, II, and III. The 
precipitate appeared to be composed largely of globulin, which in 
the state of purity obtained at the time by Felton had an isoelec- 
tric zone between 6.6 and 7.5. 

Banzhaf, 70 by Felton's method of dialysis, separated approxi- 
mately 90 per cent of the protective bodies from the fraction of 
globulins precipitable between the limits of 30 and 50 per cent satu- 
ration with ammonium sulfate. In further studies, Felton 397 " 400 ' 403 
found that the water-insoluble globulin retained its protective 
power after repeated purification, and that the globulin could be 
largely freed from accompanying serum proteins through precipi- 
tation with appropriate concentrations of sodium sulfate and by 
ethyl alcohol. 408 The possible protein nature of the immune sub- 
stance was suggested by the fact that its protective value was di- 
minished by the digestive action of pepsin, trypsin, pancreatin, 
and papain (Felton and Kauffmann, 1927). 426 

Goodner, 528 by the addition of appropriate amounts of water, 
was able to precipitate the protective antibody as well as agglu- 
tinins from antipneumococcic serum. The dilution of serum, after 
preliminary tests to determine the proper amount of water to be 
added, was carried out at a temperature of approximately 4°. 
After carrying the precipitate through a refining process, the re- 



ANTIBODIES TO PNEUMOCOCCUS 413 

suiting solution was said to contain 14,000 Felton protective units 
per cubic centimeter. 

The question arose whether the protective principle against 
Pneumococcus in specific immune serum is actually a protein of 
the globulin type representing merely an increase in the native se- 
rum globulin, whether it is a new protein substance associated with 
but chemically distinct from the natural globulin of serum, or 
whether it is a non-protein substance sharing some of the physical 
and chemical properties of serum pseudoglobulin. From time to time 
various answers have been proposed. The work of Avery, of Banz- 
haf, and of Felton indicated that the protective antibody is inti- 
mately associated with the pseudoglobulin fraction of immune se- 
rum, but the results do not necessarily imply that the antibody is 
the natural serum globulin. For example, the experiments of Fel- 
ton and Kauffmann showed that the ratio of protein to protective 
units can be reduced by selective salting out with ammonium sul- 
fate. A difference between immune and native serum globulin is the 
more alkaline isoelectric point of water-insoluble globulin contain- 
ing protective antibody, as shown by Felton and by Reiner and 
Reiner. 1133 The unusual alkaline isoelectric point of the purified 
immune proteins was considered by Chow and Goebel 226 to be its 
most characteristic property, and the authors believed that this 
property might be attributed to the relatively high ratio of amino 
to carboxyl groups present in the protein molecule. 

Chow and Goebel by repeatedly precipitating pneumococcal an- 
tibody with ammonium sulfate were able to rid the specific pseudo- 
globulin from much inert protein material, and then by means of 
potassium acid phthalate they succeeded in effecting a further 
purification of the pseudoglobulin antibody. Although Huntoon 666 
believed that the specific protective antibody separated by him 
from pneumococcal antigen-antibody precipitates was non-protein 
in nature, Chow and Goebel were of the opinion that the circulat- 
ing antibodies are in reality modified serum globulins. Confirma- 
tion of the latter hypothesis has come from the work of Heidel- 



414 BIOLOGY OF PNEUMOCOCCUS 

berger and Kendall, 625 who, by the use of strong salt concentration 
and proper adjustment of the hydrogen ion concentration well on 
the alkaline side, obtained from pneumococcal polysaccharide- 
homologous antiserum precipitates a protein substance of which 
93 per cent of the total nitrogen was in the form of immune body 
nitrogen. 

Heidelberger and Kendall took the precaution of removing from 
their source material — unconcentrated Type I, II, and III anti- 
pneumococcic serum — somatic protein antibodies and somatic car- 
bohydrate (Fraction C) antibodies, thus reducing the antibody 
content of the serum to antipolysaccharide antibodies. By employ- 
ing unconcentrated serum without chemical treatment, any pos- 
sible denaturation of the antibody was avoided and the antibody 
dissociated from the specific precipitate was considered as being 
presumably free from artificial concomitants. 

By dissociating antibody from similar antigen-antibody precipi- 
tates and then by purifying the antibody by dialysis with subse- 
quent precipitation at its isoelectric point, Chow and Wu 227 suc- 
ceeded in obtaining a protein preparation of high precipitating 
activity, which they considered as representing an immunologically 
pure protein. 

The work of Wyckoff 1555 and of Heidelberger, Pedersen, and 
Tiselius 629 further attests the protein nature of pneumococcal an- 
tibody and points to a distinction between specific immune globu- 
lin and the globulin of normal horse serum. However, there still 
remain discrepancies to be explained. None of the antibody prepa- 
rations so far obtained can be regarded as pure in the strict sense 
of the word from either a chemical or an immunological viewpoint. 
As Chow and Goebel remarked, no end-product ever attains a value 
of 100 per cent of type-specific precipitable protein. Yet the evi- 
dence that has accumulated in the past few years makes it difficult 
to escape the conviction that pneumococcal antibody is a protein, 
undoubtedly of the pseudoglobulin type, and that it is a chemical 
entity distinct from native serum pseudoglobulin. 



ANTIBODIES TO PNEUMOCOCCUS 415 

Further information concerning the intimacy existing between 
pneumococcal antibody and immune serum globulin has come from 
analyses of normal and antipneumococcic serum by means of the 
ultracentrifuge of Svedberg 1369 and a modified machine described 
by Biscoe, Pickels, and Wyckoff (1936). 119 Employing the latter 
apparatus, Biscoe, Hercik, and Wyckoff 118 determined that in con- 
centrates of Type I antibodies the proteins consist mainly of mole- 
cules with a sedimentation constant of about 

16 X 10 13 cm. sec" 1 dynes" 1 . 

This finding, taken with the presence of similar molecules in uncon- 
centrated antipneumococcic horse serum and their complete or al- 
most complete absence from normal serum indicates that these 
molecules are the real bearers of antibody activity. Further sup- 
port of the view is supplied by the observation of Heidelberger, 
Pedersen, and Tiselius 629 that after ultracentrifugation the spe- 
cific antibody from serum from a horse immunized with Type I 
polysaccharide and a sample of concentrated Type I antipneumo- 
coccic serum (Felton) showed homogeneous sedimentation, with 
the following sedimentation constant: 

* 20 = 17.2 X 10 13 

The sedimentation constant of normal globulin from mammalian 
serum is: 

s 20 = about 7 X 10" 13 

By applying the method to concentrated preparations of Type 
I antipneumococcic serum, Wyckoff 1555 found that the only pro- 
tein molecules in the bottom layers of the fluid were those with 

s= 16 X 10 13 

Small amounts of light molecules, which in each concentrate ac- 
counted for roughly 15 per cent of the total protein content, were 
those of the principal serum globulin with 

s = 7X 10 13 



416 BIOLOGY OF PNEUMOCOCCUS 

The experiments strongly support the idea that the Type I anti- 
body is associated with the molecule having 

s=16X 10 13 

Furthermore, Biscoe, Pickels, and Wyckoff obtained the same 
measurement for molecules of antibodies against other types of 
pneumococci. Speculating on the relation between pneumococcal 
antibody and specific immune protein, Wyckoff concluded: "Since 
this is a molecular species that appears in appreciable quantities in 
horse serum only when it has become antipneumococcic, two possi- 
bilities suggest themselves. It is conceivable that during immu- 
nization this globulin is made or freed in excess in order that there 
may be plenty of it present to fix all the antibody activity that 
may develop . . . and the alternate, that the globulin is the anti- 
body." The results of investigations of this nature are highly sug- 
gestive, and the method promises to yield valuable information 
concerning this abstruse but important question. 

Differences in the nature of the protective antibody in anti- 
pneumococcic rabbit serum and in that in antipneumococcic horse 
serum have been demonstrated by Chow. 224 In horse serum, the an- 
tibody in the pseudoglobulin fraction soluble at pH 5.5 was com- 
pletely precipitated when the reaction of the solution was ad- 
justed to pH 7.6. From the corresponding fraction of the immune 
rabbit serum no trace of precipitate was obtained. Furthermore, 
in the case of immune horse serum, the main portion of the anti- 
body was concentrated in the pseudoglobulin fraction, whereas the 
opposite was found to be true with the immune rabbit serum, that 
is, the euglobulin fraction apparently contained the major portion 
of the antibody.* 

Another difference in the antibodies present in antipneumococcic 
rabbit and horse serum lies in the size of the molecule of the two 
substances. In experiments involving the use of the ultracentri- 

* A difference in the phosphatid molecules linked with the globulin in the 
case of antibodies from the horse and rabbit has been mentioned in the foot- 
note on page 381 of this chapter. 



ANTIBODIES TO PNEUMOCOCCUS 417 

fuge, Heidelberger, Pedersen, and Tiselius observed that in the 
case of immune rabbit serum the specific antibody was produced 
from the principal globulin component, while in the horse the anti- 
carbohydrate for Type I Pneumococcus is developed from an 
otherwise minor component. The isoelectric point of the protein 
representing the pneumococcal antibody of the rabbit was deter- 
mined as approximately pH 6.6, whereas the isoelectric point of 
the immune protein from horse serum was as acid as pH 4.8. That 
the molecule of the protective antibodies as elaborated by the two 
animal species is of different size is also evident from the work of 
Goodner, Horsfall, and Bauer. 689 When Type I antipneumococcic 
rabbit serum was filtered through an ultrafilter of the type de- 
scribed by Bauer and Hughes, 90 no appreciable specifically pre- 
cipitable protein passed through a membrane with an average pore 
diameter of one millimicron. A 13.8 millimicron filtrate con- 
tained 11.9 per cent of the total specifically precipitable protein. 
Slightly greater amounts were recovered as the pore sizes were in- 
creased up to 73 millimicrons, at which point the curve rose 
sharply until at 102.5 millimicrons the filtrate contained 86.6 per 
cent of the total antibody. With antipneumococcic horse serum, 
the smallest pore permitting the passage of antibody was 45.2 
millimicrons. Between 73 and 102.5 millimicrons the curve rose 
steeply until at the latter porosity 76.7 per cent of the antibody 
was recovered. With a concentrated preparation of Type I anti- 
pneumococcic horse serum the end-point was relatively sharp, no 
antibody being recovered at 150.4 millimicrons, while 100 per 
cent was found in the filtrate at 188 millimicrons. The authors 
decided that in general it might be assumed that the smallest spe- 
cific antibody of antipneumococcic rabbit serum corresponds to a 
pore size of 11 millimicrons, the smallest in horse serum to a size 
of 44 millimicrons, while both horse and rabbit antibodies have 
large specific aggregates corresponding roughly to a pore size of 
88 millimicrons. Furthermore, the antibody of concentrated horse 
serum requires a pore size of approximately 176 millimicrons. 



418 BIOLOGY OF PNEUMOCOCCUS 

Goodner, Horsfall, and Bauer drew attention to the fact that the 
figures given are multiples of 11, which corresponds to the value 
of 11 to 12 millimicrons determined by Elford and Ferry 353 for 
the isolated form of normal horse pseudoglobulin. 

Recent observations on the molecular size of pneumococcal anti- 
body in immune rabbit serum, in immune horse serum, and in con- 
centrated antibody solution are of importance not only in con- 
nection with differences in the characters of antibody as produced 
in animals of different species, but in emphasizing the range in sur- 
face area of the different types of antibody. Gram for gram, the 
pneumococcal antibody from the immune rabbit, because of its 
smaller molecular size and therefore its greater surface area, 
should exhibit greater combining power for homologous antigen 
than the antibody of the larger molecule of immune horse serum 
and that of the still larger aggregate present in concentrated anti- 
pneumococcic serum. 

THE SEPARATION OF ANTIBODIES FROM IMMUNE SERUM 

Inasmuch as the concentration and purification of pneumococ- 
cal antibodies will be described in detail in Chapter XV, only the 
principles involved in the separation of specific substances from 
immune serum will be dealt with in the present chapter. Gay and 
Chickering 508 " 9 took advantage of the particulation that takes 
place when cellular substances of Pneumococcus are brought into 
contact with homologous immune serum. From the antigen-anti- 
body precipitates the antibody was then recovered by the use of 
suitable physical and chemical agents. The method was adopted, 
with modifications, by Huntoon and his associates 665 " 6 ' 668 " 9 and the 
use of specific precipitates has been developed as furnishing a 
source of pneumococcal antibody. Another basic method is founded 
on the relative insolubility of the immune proteins in water at their 
isoelectric point, as well as their insolubility in water in the ab- 
sence of electrolytes. The use of protein coagulants such as am- 
monium and sodium sulfates, so valuable in the concentration and 



ANTIBODIES TO PNEUMOCOCCUS 419 

refinement of antitoxins, and a similar use of alcohol, have re- 
ceived wide application in the study of the chemical nature of im- 
mune substances against Pneumococcus and especially in the quan- 
tity concentration of antipneumococcic serum. The affinity of the 
immune bodies for metallic salts also affords a method for the iso- 
lation of antibody. Now ultracentrifugation and ultrafiltration 
promise means of separating pneumococcal antibody from the ac- 
companying components of immune serum. 

THE ESTIMATION OF PROTECTIVE ANTIBODIES 

In the quantitative estimation of the content of protective anti- 
body in antipneumococcic serum several variables are encountered 
in the culture, the serum, and the test animal employed. Inasmuch 
as methods for measuring the therapeutic potency of antipneumo- 
coccic serum will be discussed in the chapter dealing with serum 
production, brief mention may be made at this point of some of the 
factors which may influence the accuracy of the determination. 
Enlows 366 pointed out the necessity of using cultures in the phase 
of active growth or in the early stationary phase in order to avoid 
lag on transfer. The hydrogen ion concentration of the medium in 
which the pneumococci are grown should be such as to ensure the 
presence of cocci at the height of vitality. The virulence of the 
culture should be maintained at a high level. Felton 402 standard- 
ized the infecting dose of culture by plate counts and employed a 
dilution of culture representing 500,000 fatal doses of organisms. 

Felton (1926) 399 discovered in antipneumococcic serums for 
Types I, II, and III, a substance that was lethal for mice. Later, 
Felton and Bailey 419 pointed out that from immune serum precipi- 
table residues could be isolated that had an antagonistic action on 
the neutralization of protective antibody by capsular polysaccha- 
ride. Furthermore, the amount of soluble specific substance oper- 
ated to cause a zonal effect in the estimation of protective action. 

The mouse, on account of race, age, weight, and physical condi- 
tion, presents an array of variable factors in the response to the 



420 BIOLOGY OF PNEUMOCOCCUS 

protective action of specific antipneumococcic serum. These fac- 
tors, described by Felton 405 for the mouse, and by Goodner, 532 " 4 
and Goodner and Miller 540 for the mouse and rabbit, have already 
been mentioned in the chapter on antigenicity and need not be 
repeated. 

For elimination of the variables in the animal host, for sim- 
plicity and rapidity of performance, the newer methods based on 
specific precipitation present many advantages over the mouse 
protection test for the quantitative determination of protective 
antibody in antipneumococcic serum. 

Other Immunological Phenomena 

GROWTH OF PNEUMOCOCCI IN SPECIFIC IMMUNE SERUM 

The growth of pneumococci in immune serum was observed by 
Metchnikoff 894 in 1891, and Denys (1897) 312 reported that pneu- 
mococci grow as well in immune as in normal rabbit serum. In the 
experiments of Rosenow (1904), 1159 pneumonic blood was found to 
possess no bactericidal properties for Pneumococcus, while viable 
organisms could be propagated from agglutinated masses of the 
cocci in the serum of pneumonia patients. An initial lag in the mul- 
tiplication of pneumococci planted in the serum of artificially im- 
munized animals, followed by active proliferation of the cells, was 
described in 1920 by Bull and Bartual. 177 Nicolle and Cesari 
(1926) 1008 obtained better growth in Martin bouillon containing 
specific homologous serum than in the same medium to which het- 
erologous immune or normal serum had been added. The ability of 
pneumococci to grow in homologous anti-S and anti-R immune se- 
rum and the effects of the serum on the growth of the cocci has 
been described in Chapter V. 

ANTIBLASTIC IMMUNITY 

An action of antipneumococcic serum apparently not definitely 
attributable to any of the known specific antibodies was described 



ANTIBODIES TO PNEUMOCOCCUS 421 

in 1916 by Dochez and Avery, 320 who found that immune serum 
temporarily inhibited the multiplication of pneumococci and, at 
the same time, depressed the proteolytic and glycolytic functions 
of the bacterial cell. The retardation of growth and inhibition of 
metabolic activity of the cocci was ascribed by the authors to anti- 
enzymatic substances in antipneumococcic serum, and to the phe- 
nomenon they applied the term "antiblastic immunity." Blake 
(1917) 124 differed with Dochez and Avery in the explanation of the 
effect, and since it was found that the inhibitory action of serum 
paralleled the agglutinative power and, moreover, since serum ex- 
hausted of its agglutinins no longer interfered with the metabolic 
activities of the cell, Blake denied the participation of any anti- 
enzymatic principle in the phenomena. Barber (1919) 78 observed 
the inhibitory effect on the vital activities of pneumococci of whole 
fresh blood, coagulated plasma, and the serum of normal or im- 
mune horses and pigeons and, while attempting no analysis of the 
factors involved, believed that the action was antiblastic in na- 
ture. Bordet (1931 ), 140 noting the effect of normal rabbit serum 
on cultures of Pneumococcus, as manifested by changes in the 
morphology of the cocci, queried whether the disturbance in the 
metabolism of the organisms could be due to alexin or to the pro- 
duction of acid in cultures containing rabbit serum. The influ- 
ences displayed by immune serum on the vital functions of pneu- 
mococci, as just described, are probably the same as those leading 
to the dissociation and degeneration of the pneumococcal cell. 

IMMUNOLOGICAL RELATIONSHIPS BETWEEN PNEUMOCOCCUS AND 
OTHER MICROBIC SPECIES AND UNRELATED SUBSTANCES 

Analogous to the apparent serological relations already de- 
scribed in Chapter VIII existing between Pneumococcus and such 
varied entities as gum arabic, Bacillus coli, Leuconostoc mesen- 
teroides, Friedlander's bacilli, and tubercle bacilli, are the cross- 
reactions demonstrated by Sugg and Neill (1929) 1855 between 
yeast and Type II Pneumococcus. The reader may recall that 



422 BIOLOGY OF PNEUMOCOCCUS 

Mueller and Tomcsik (1924) 938 had described a complex carbohy- 
drate prepared from yeast that bore certain chemical resemblances 
to the capsular polysaccharide of Pneumococcus. It remained for 
Sugg and Neill 1356 to demonstrate that the resemblance extended 
to the immunological behavior of these two representatives of 
Schizomycetes and Saccharomycetes. Pneumococci of Type II, but 
not of Types I or III, were agglutinated by serum from rabbits 
immunized by injections of a certain variety of yeast, and filtrates 
of young, unautolyzed broth cultures of the same type of pneumo- 
cocci invariably precipitated potent antiyeast serum. The anti- 
yeast serum, furthermore, protected normal mice against Type II 
pneumococci as well as the average specific antiserum produced in 
rabbits for this type. The reciprocal reaction of Type II anti- 
pneumococcic serum with yeast was not so definite, since serum from 
many normal rabbits was capable of agglutinating yeast. In a 1 to 
5 dilution, Type II antipneumococcic serum gave clean-cut agglu- 
tination. The results of absorption experiments with both the anti- 
yeast (rabbit) serum and the Type II antipneumococcic (horse) 
serum were the same as those usually obtained in analogous experi- 
ments with immunologically related, but not identical, species of 
bacteria. 

The protection against infection with Type II pneumococci con- 
ferred upon mice by injection of suspensions of heated yeast cells 
was practically the same as that obtained by vaccination with 
Type II pneumococci themselves, with the exception that yeast 
evoked immunity to Type II and not to Type I or Type III or- 
ganisms. The mutual reactions of yeast and Type II pneumococci 
were further demonstrated when Sugg and Neill (1931) 1356 em- 
ployed semi-purified carbohydrate antigens from both yeast cell 
and pneumococci mixed with homologous antiserums. Antiyeast 
serum precipitated with, and sensitized guinea pigs to the Type II 
antigen but was not active with antigens from strains of Type I 
and III pneumococci. The Type II antigen was almost as reactive 
against antiyeast serum as against homologous antiserum. Con- 



ANTIBODIES TO PNEUMOCOCCUS 423 

versely, Type II but not Type I or Type III antipneumococcic se- 
rum reacted with yeast carbohydrate antigen but not to the degree 
observed in the case of antiyeast serum and pneumococcal carbo- 
hydrate. The action of different samples of antiyeast serum of 
equal potency as to reactivity with yeast antigen varied greatly 
when tested against pneumococcal antigen. 

Summary 

The substance of this chapter may be recapitulated as follows : 
The introduction into the bodies of animals of suitable species of 
pneumococci and some of their natural components augments or 
brings into being an array of specific immune substances that serve 
to protect the animal against the invading cocci and that can be 
demonstrated by appropriate serological and other immunological 
reactions. The immune substances thus evoked comprise agglu- 
tinins, precipitins, opsonins or tropins, and complement-fixing and 
protective antibodies. 

Agglutinins, arising as a result of artificial immunization or ap- 
pearing in the blood of pneumonia patients at or near the time of 
crisis, are easily demonstrated : the specific agglutination reaction 
takes place between the intact pneumococcal cells, whether living 
or dead, and the homologous antibody in the serum used. The re- 
action varies from strict type-specificity to a broader species- 
specificity, depending upon the nature of the antigen employed in 
the production of the immune serum and that of the hapten par- 
ticipating in the reaction. The somatic protein of the cocci and of 
degraded or R forms of the organisms engenders agglutinins reac- 
tive with all types of pneumococci, whereas the intact, virulent 
forms of pneumococci or their unimpaired capsular constituents 
evoke agglutinative substances strictly specific for the type of anti- 
gen or component supplying the antigenic stimulus. In the latter 
instance the reaction is one between the specific capsular polysac- 
charide of the organism and its corresponding specific, immune- 
serum globulin. Because of the simplicity of the technique required 



424 BIOLOGY OF PNEUMOCOCCUS 

for the detection or quantitative determination of agglutinins in 
immune serum, the agglutination reaction finds a wide use in the 
identification of members of the types within the species. 

The precipitin reaction, involving as it does the interaction of 
hapten and immune body in solution instead of the reaction be- 
tween formed microbic elements and specific serum and, moreover, 
being independent of the participation of the immunological fac- 
tors possessed by a living, immune animal, is susceptible to the 
application of mathematically accurate quantitative measure- 
ments. By means of the precipitin reaction, it is possible to deter- 
mine with great exactness the quantity of hapten in a given solu- 
tion or the amount of immune nitrogen — that is, the amount of 
specific antibody in immune serum. Because of the fortunate, close 
parallelism existing between the content of precipitin and protec- 
tive antibody in any particular serum, a quantitative estimation 
of the therapeutic value of antipneumococcic serum can be made 
in the test tube instead of in experimental animals. 

The greater economy of means required in the reaction and its 
greater accuracy when properly performed have led to far more 
detailed studies of the phenomenon which, in turn, have supplied 
many clues to the mechanism operating in immunity to Pneumo- 
coccus. The chemist has thus been given the means of learning the 
very chemical radicals in the precipitinogen and the globulin frac- 
tion of immune serum which join to produce the precipitate and 
presumably which participate in the other immunological manifes- 
tations. The specificity of serological precipitation depends on the 
interaction of the capsular polysaccharide and immune globulin; 
and the basic process is the same as that in the phenomenon of 
agglutination. 

The method of complement fixation offers no advantages over 
other serological methods for the demonstration or measurement 
of pneumococcal antigen and antibody. It has, however, revealed 
and enabled one to study the striking difference in the nature of 
specific antibodies in immune rabbit serum and immune horse serum. 



ANTIBODIES TO PNEUMOCOCCUS 425 

Substances that neutralize the hemotoxin of Pneumococcus have 
been discovered in specially prepared immune serum, and some evi- 
dence has been presented that substances capable of inhibiting the 
action of the poisonous products of Pneumococcus may arise in 
the immunization of animals with the so-called pneumococcal 
toxins. 

In a true sense there are no specific bactericidins for pneumo- 
cocci. The destructive effect on pneumococci of body tissues is ref- 
erable to a more complex play of biological elements. 

There exist in the normal animal body, and to a much greater 
degree in the body of immune animals, substances that render pneu- 
mococci susceptible to the phagocyting action of mobile leucocytes 
and the fixed body cells. Disregarding the confusion of terms, the 
opsonins or tropins act on the bacteria and not on the phagocytic 
cells and it is by means of the intervention of the normal or im- 
mune opsonins that, under suitable conditions, pneumococci may 
be destroyed in the test tube or in the body of the infected animal. 

The combined phenomena of agglutination, precipitation, and 
opsonization are the complement of forces mustered by the animal 
body for protection against invading pneumococci. The assump- 
tion is supported by the close correlation found to exist between 
the various antibodies concerned in antipneumococcal immunity. 
The specific protective action of immune serum can be strikingly 
demonstrated in laboratory animals and the type-specificity and 
the degree of this protective action can be measured by in vitro 
methods. Through chemical studies, the protective antibodies have 
been located in the globulin fraction of immune serum and by both 
chemical and physical manipulations they may be isolated in rela- 
tively pure form. Recent advances in these technical procedures 
have made possible actual measurements of the molecular size of 
pneumococcal antibodies and are leading to a clearer conception of 
their chemical composition. 

There remains to be explained the significance of the immuno- 
logical relation of pneumococcal antigens and antibodies to those 



426 BIOLOGY OF PNEUMOCOCCUS 

of alien microbic species, as well as their relation to apparently 
biologically unrelated substances in both animal and vegetable tis- 
sues. The solution of these problems will go far to explain depend- 
ence of immunological action on chemical composition. 



CHAPTER XII 

HOST RESPONSE TO ANTIGENIC ACTION 
OF PNEUMOCOCCUS 

Normal barriers to the invasion and multiplication of pneumo- 
cocci in the animal body; the immunological reaction naturally 
stimulated by pneumococcal infection and the reaction artificially 
aroused by the administration of pneumococci, their constituents, 
or derivatives; the somatic manifestations of the immune state; 
and the utilization by the body of antibodies passively acquired. 

Sound skin and healthy mucous membranes are impediments to 
the entrance of Pneumococcus into the animal body. Under- 
lying these tissues, as a line of secondary defense, are the leuco- 
cytes, which with normal auxiliary elements of the blood may in- 
gest and destroy the invading cocci. The localization or the sys- 
temic distribution of the organisms depends on the vigor of the 
phagocytic response and on the functional capacity of the body 
to elaborate specific antibodies in response to the antigenic stimu- 
lus provided by the invading microorganisms. 

Total failure of the defenses means death but, with only a par- 
tial lack of antagonistic factors, the animal may pass through the 
rigors of pneumococcal disease and thereby acquire greater, if 
temporary, resistance to subsequent attacks. An analysis of the 
protective mechanism of the animal economy against pneumo- 
coccal infection will be attempted in the present chapter. 

Natural Immunity 

IN ANIMALS 

A reason for the behavior of various normal animals toward 
pneumococcal infection was sought by Tchistovitch (1890), 1381 
who subjected dogs, rabbits, and mice to subcutaneous, intra- 
tracheal, and intraocular inoculation with Streptococcus lanceo- 



428 BIOLOGY OF PNEUMOCOCCUS 

latus (Pneumococcus). The animals were killed during successive 
stages of infection and the lungs examined for cellular changes. 
The differences in the physiological response corresponded with 
the natural susceptibility of the animal species to infection. In 
non-refractory animals, the cocci caused only a feeble local in- 
flammatory reaction with little phagocytosis, and the leucocytes 
neither engulfed the organisms nor inhibited their growth. In re- 
fractory animals, on the contrary, a more or less pronounced 
local inflammatory process developed, with leucocytic migration 
and accompanying phagocytosis. When the injections were made 
into the anterior chamber of the eye, there appeared to be no dif- 
ference in the reaction of the aqueous humor of the animals of the 
two classes, since the fluid served as a medium for the growth of 
the injected cocci. 

Opposed to the observations of Tchistovitch were those of Behr- 
ing and Nissen, 98 published in the same year, who could find no 
differences in the bactericidal properties of the serum of mice, rats, 
and rabbits. The inability of the serum of the animals to affect 
pneumococci was in marked contrast to their destructive action on 
anthrax bacilli. Wadsworth 1455 also (1903) was unable to discover 
any parallelism between the action on pneumococci of serum from 
normal animals and the natural resistance of the animals to pneu- 
mococcal infection. When tested by agglutination and precipita- 
tion methods, the serums in low dilutions showed no significant 
differences. 

Natural immunity of the pigeon to Pneumococcus, according to 
Strouse (1909), 1345 was due to high normal body temperature and 
not to any specific tissue reaction. Employing both in vitro and 
in vivo methods for studying phagocytosis, Strouse could detect 
no difference in the reaction of pigeons and of mice to experi- 
mental infection. Ungermann 1434 also studied the phenomenon of 
phagocytosis occurring after the addition of serum of various 
animal species to leucocytes of animals of homologous and heter- 



HOST RESPONSE TO ANTIGENIC ACTION 429 

ologous species. While in the case of the rabbit and mouse no 
phagocytosis was observed when virulent pneumococci were used 
in the test, the destruction of avirulent strains seemed to parallel 
the resistance toward the particular strain possessed by the ani- 
mal whose serum was tested. While Dold (1911) 323 could demon- 
strate no substances antagonistic to Pneumococcus in the serum, 
plasma, or whole blood of normal mice and rabbits, by treating 
rabbit leucocytes by the method of Schneider, extracts were ob- 
tained which Dold claimed had definite killing power for pneu- 
mococci. 

Robertson and Sia (1923) 1144 devised an accurate method for 
demonstrating growth-inhibitory and bactericidal action on Pneu- 
mococcus of normal serum-leucocyte mixtures. The ingredients of 
the mixture were added in known quantities and mechanically agi- 
tated by rotation and oscillation. A combination of serum and 
leucocytes from resistant animals (cats and dogs) exerted not 
only a growth-inhibiting but also a bactericidal action on pneu- 
mococci. The serum-leucocyte mixtures of susceptible animals 
(rabbits and guinea pigs) showed no inhibitory effect. In subse- 
quent communications, Robertson and Sia 1145 " 7 substantiated their 
earlier results. For example, the growth of pneumococci possess- 
ing low virulence for the cat was found to be markedly inhibited 
in mixtures of cat serum and cat leucocytes, since ten thousand 
times the number of pneumococci ordinarily sufficient to kill a 
mouse failed to infect after being exposed for twenty-four hours 
in the cat serum-leucocyte mixtures. Furthermore, virulent strains 
sensitized by contact with serum of animals resistant to Pneumo- 
coccus were actively phagocyted, not only by the homologous leu- 
cocytes, but also by the leucocytes of other resistant animals and 
of susceptible animals. However, pneumococci exposed to the ac- 
tion of serum from susceptible animals were not taken up by leu- 
cocytes of either the resistant or susceptible animals. The serum 
of all the resistant animals tested — dog, cat, sheep, pig, and horse 



430 BIOLOGY OF PNEUMOCOCCUS 

— showed marked opsonic properties which were absent from the 
serum of animals of such susceptible species as the rabbit, guinea 
pig, and man. In contrast to the activity of serum there appeared 
to be no essential difference in the phagocytic power of the leuco- 
cytes from the various animals. The influence of the age of the ani- 
mals on the pneumococcidal properties of the blood was shown by 
the fact that mixtures of adult rabbit serum with either adult or 
young hare leucocytes exerted pronounced growth-inhibitory and 
coccidal action, whereas mixtures of serum from immature hares 
with leucocytes from either adult or young rabbits completely 
lacked any similar action. In the natural defense mechanism 
against pneumococcal infection, it appears that the serum con- 
tains the potential elements and that in a susceptible animal like 
the rabbit the elements develop with the growth of the animal. 

The results reported by Robertson and Sia were practically 
duplicated by those of Woo, 1541 who found that rabbit serum-leu- 
cocyte mixtures possessed the power to kill avirulent pneumococci 
in relatively large numbers, but failed to inhibit growth of even 
minute quantities of virulent organisms, an observation also re- 
ported by Wright (1927). 154T Woo found further that the serum 
of very young animals when mixed with leucocytes was powerless 
to affect cultures which were without virulence for mature rab- 
bits. Bull and Tao (1927) 182 introduced citration for determining 
the antipneumococcic properties of whole blood. One per cent by 
volume of saturated, neutral sodium citrate delayed coagulation 
of the blood for twenty-four hours and did not inhibit the growth 
of pneumococci. The killing action of citrated blood was more po- 
tent than that of serum-leucocyte mixtures. When the method was 
applied to the blood of normal rabbits and chickens, Bull noted 
that it required one million times as many pneumococci to infect a 
given quantity of chicken blood as it did to infect the same volume 
of rabbit blood. 

The hypothesis that it was the natural, humoral antibodies 



HOST RESPONSE TO ANTIGENIC ACTION 431 

which were responsible for the pneumococcidal action of serum- 
leucocyte mixtures received confirmation in a separate study by 
Sia (1927), 1268 in which it appeared that the active principle op- 
erative in the destruction of pneumococci in appropriate serum- 
leucocyte mixtures could be specifically absorbed from the serum, 
and that the native principle — the opsonin — was type-specific in 
its selective action. 

It was found by Kelley (1932), 701 as Robertson and Sia had 
previously discovered, that normal swine serum possessed the 
property of protecting mice against infection with virulent pneu- 
mococci and of agglutinating both virulent smooth and avirulent 
rough strains of the organism. The protective action of swine 
serum, although slight, was evidently specific for type, since the 
protective antibody for one type of Pneumococcus could be ab- 
sorbed by cultures of the homologous type without affecting the 
content of antibody for other types. Unlike the protective sub- 
stances in specific immune serum, those in normal pig serum were 
thermolabile and disappeared after a few weeks' storage in the 
cold and, moreover, they could be removed from the serum by ab- 
sorption with avirulent rough pneumococci. Another difference be- 
tween normal swine serum and specific immune serum lay in the 
fact that although the former was type-specific in mouse-protec- 
tive action, when mixed with soluble specific substance no precipi- 
tation took place, nor was the protective action inhibited by the 
carbohydrate. Kelley also confirmed Robertson and Sia's observa- 
tion that swine serum shows type-specific agglutination for pneu- 
mococci, and that agglutinins for one type may be specifically ab- 
sorbed, leaving agglutinins for other types undisturbed. Inasmuch 
as normal pig serum agglutinates avirulent, rough forms of pneu- 
mococci and the agglutinative property is not destroyed by heat- 
ing at 56°, Kelley assumed that this property probably depends 
on factors other than those responsible for agglutination of 
smooth forms of Pneumococcus and for protective action in mice. 



432 BIOLOGY OF PNEUMOCOCCUS 

In 1929, Sia 1269 presented further evidence in support of the 
view that humoral defensive elements play an important part in 
natural immunity to Pneumococcus. Mice were given intraperi- 
toneal injections of serum from the pig and four hours later ac- 
tively growing cultures of virulent Type I pneumococci were in- 
jected into the animals. Control mice were similarly injected with 
serum from susceptible animals (the rabbit and guinea pig) and 
then inoculated with the same culture. Normal swine serum pro- 
tected the mice against ten thousand minimal lethal doses of cul- 
ture, whereas serum from the susceptible animals afforded no pro- 
tection to the test animals. Pig serum also protected mice against 
virulent Type II pneumococci and to a lesser degree against Type 
III organisms. Moreover, absorption experiments again demon- 
strated that the protective substance was type-specific. 

Le Guyon (1931) 796 investigated the causes of the different re- 
actions exhibited by rabbits and guinea pigs to pneumococcal 
infection. The technique employed consisted in inoculating the 
animals intraperitoneally with a broth culture of virulent pneu- 
mococci and in examining peritoneal fluid withdrawn at various 
intervals after injection. Four hours after inoculation, mice de- 
veloped a marked cellular reaction. Along with many cocci there 
were always a large number of macrophages of the clasmatocyte 
type. There was intense phagocytosis by the polymorphonuclear 
leucocytes and macrophages, but in spite of the reaction the ani- 
mals developed septicemia and died within eighteen to twenty 
hours. In guinea pigs four hours after inoculation, the reaction 
was feeble. Some polymorphonuclear cells and some free pneumo- 
cocci were present in the exudate, but there was only occasional 
evidence of phagocytosis. Sixteen hours later, the number of or- 
ganisms and polymorphonuclear leucocytes had greatly increased, 
but lymphocytes and macrophages were rarely seen. The pneu- 
mococci, some of which were phagocyted, were clustered about 
the polymorphonuclear cells and appeared to be in a degenerated 
condition. Le Guyon therefore concluded that the initial refrac- 



HOST RESPONSE TO ANTIGENIC ACTION 433 

tory state in the guinea pig was due to the bactericidal property 
of the serum. 

Bordet (1933) 141 believed that in addition to the leucocyte- 
serum complex there were other humoral factors which accounted 
for the unequal susceptibility of animals of different species. The 
action of the hypothetical factors was manifested by the altered 
appearance of pneumococci grown in the serum of different ani- 
mals. The principles appeared to be thermolabile, could be ab- 
sorbed by aluminum hydroxide, and accompanied the euglobulin 
when serum was separated into its protein fractions. 

A type of resistance, quite independent of strictly immunologi- 
cal processes, is that possessed by the rabbit against the majority 
of strains of Type III Pneumococcus. An experimental infection 
can be induced in the rabbit by intradermal injection of many 
strains of this serological type but the infective process is aborted 
and the animal recovers. 

In the early stages of the infection the leucocytes are powerless 
to engulf the cocci but, as the body temperature of the animal 
rises to 104° or higher as a result of the infection, the organisms 
lose their capsules and are avidly phagocyted. One of the deter- 
mining factors in the inability of many Type III pneumococci to 
produce fatal infection in the rabbit is the susceptibility of the 
organisms to the high temperature generated in the rabbit. A de- 
tailed discussion of the experimental evidence on which the above 
statement is based is to be found on pages 207 to 210 in Chap- 
ter VI. 

IN MAN 

The presence of normal precipitin for Pneumococcus in the 
blood serum of man was reported by Wadsworth (1903) 1455 but its 
content was low, since in serum in a dilution of 1 to 10 or more the 
action of the native precipitin was not evident. Rosenow (1904) 1159 
denied that fresh normal blood or serum had any bactericidal in- 
fluence on Pneumococcus. Neufeld and Haendel (1910) 990 demon- 



434 BIOLOGY OF PNEUMOCOCCUS 

strated the protective action of normal human serum for the mouse, 
while Much* reported that normal human serum, and to a still 
higher degree, plasma, contained thermolabile bactericidal sub- 
stances for Pneumococcus. 

Dold (1911), 323 testing by means of plate cultures the action 
of serum, plasma, and whole blood of normal human beings and of 
patients ill with diseases other than pneumonia, claimed to have 
demonstrated definite pneumococcidal effect. The action, stronger 
in blood than in plasma or in serum, varied from slight inhibition 
of bacterial growth to actual killing of the organisms. 

According to Clough (1924), 243 normal human beings may pos- 
sess in their serum substances capable of protecting mice against 
infection with pneumococci of Types I, II, and III. The degree of 
protection, when demonstrable, was usually slight, but in five in- 
stances was sufficient to save the animal from subsequent inocula- 
tion with 1,000 to 100,000 minimal fatal doses of pneumococci. 
Protective action against one type of organism was not neces- 
sarily accompanied by similar action against representatives of 
other types, nor was the property associated with the presence of 
any agglutinins or opsonins, since the latter antibodies were ab- 
sent from the serums tested. 

Burhans and Gerstenberger (1924) 190 tested the serum of in- 
fants and maternal parents for protective power against Type I, 
II, and III strains of Pneumococcus. The serum of approximately 
40 per cent of the parturient mothers protected mice against 
inoculation with organisms of the three fixed types, while samples 
of serum from only about 30 per cent of the infants exhibited 
similar properties. The authors decided that the low incidence of 
lobar pneumonia during infancy was probably not due to an im- 
munity to the fixed types of pneumococci. 

Ash and Solis-Cohen (1929) 26 believed that differences in sus- 
ceptibility to pneumonia among human beings could be deter- 
mined by the growth-inhibitory and pneumococcidal action of 

* Quoted by Neuf eld and Schnitzer. 



HOST RESPONSE TO ANTIGENIC ACTION 435 

whole blood and, furthermore, that the reaction was dependent 
upon the presence of leucocytes. 

Analogous differences in resistance were observed by Robertson 
and Cornwell (1930). 1143 In a study of the pneumococcidal action 
of normal human serum-leucocyte mixtures for freshly isolated 
strains of pathogenic pneumococci, the authors ascertained that 
human beings as a group possess in their blood well-marked de- 
structive properties for all types of Pneumococcus studied. Indi- 
viduals, however, exhibit wide variations in reaction against differ- 
ent types, ranging from strong killing effect for organisms of one 
type to no action or slight effect against strains of another type. 
In the light of the results of previous experiments in which actual 
resistance of animals to pneumococcal infection was determined, 
Robertson and Cornwell interpreted the findings as meaning that 
human beings in general possess a considerable degree of natural 
immunity to all types of Pneumococcus, but that some individuals 
may be susceptible to one or more types and at the same time be 
resistant to other types. 

By a method employing whole blood, Ward 1480 found that the 
phagocytic titer against the first three types of pneumococci va- 
ried through a wide range in different normal human subjects. 
Similar results were obtained in the same year by Sutliff and 
Rhoades, 1363 " 4 who measured the pneumococcidal power of normal 
human blood by a modification of the method of Robertson and his 
co-workers. Whole blood, to which heparin was added in small 
amounts as an anticoagulant, was mixed with pneumococci and ro- 
tated in an apparatus devised by the authors. Parallel determina- 
tions of the protective power of the samples were carried out. The 
blood of seventeen out of twenty-seven hospital patients who had 
not had lobar pneumonia killed from one hundred to ten thousand 
virulent Type I pneumococci. When mouse-protective power and 
pneumococcidal power were compared, it was found that six sub- 
jects possessed both properties and that the serum of ten indi- 
viduals was bactericidal but not protective, while the serum of 



436 BIOLOGY OF PNEUMOCOCCUS 

three subjects exhibited neither property. In another communica- 
tion, Sutliff with Finland 1360 reported that the incidence of pneu- 
mococcidal power and of other type-specific antibodies varied with 
the age of the subject as well as with the different types of Pneu- 
mococcus. The killing action of normal human serum was most fre- 
quently observed with organisms of Type II, was rarest for Type 
I, and was intermediate for Type III strains. 

Gundel, 567 in 1932, investigated the presence of pneumococ- 
cidins, protective antibodies, and agglutinins in the blood of nor- 
mal adults and of healthy nurslings and children. The blood of 
adults frequently showed the presence of definite amounts of anti- 
bacterial antibodies, whereas no protective or agglutinative action 
was observed with the serum of new-born babies and young chil- 
dren. Gundel concluded that antibody formation began in normal 
children toward the end of the second year of life. Variations in the 
titer of humoral immunity were manifested in a third or a fourth 
of the subjects, there being an increase in the case of some indi- 
viduals and a decrease in others. In the majority of instances the 
action was specific, since the altered reactivity toward organisms 
of one type was not accompanied by a similar change toward 
other types of pneumococci. 

An experimental analysis of the factors responsible for the 
pneumococcidal action of human serum convinced Ward and En- 
ders 1484 that in normal human serum virulent pneumococci may be 
prepared for phagocytosis by two separate antibodies acting in 
conjunction with complement. One of the substances is probably 
the type-specific anticarbohydrate antibody reacting with the 
capsular polysaccharide of Pneumococcus ; the other is probably 
also a type-specific antibody, but quite distinct from the former 
and, therefore, reacting with a different antigenic constituent of 
the bacterium. In normal human serum heated to 56° the two anti- 
bodies may, after prolonged contact with the organism, promote 
phagocytosis of pneumococci without the adjuvant action of com- 
plement. While the two antibodies are equally effective in the 



HOST RESPONSE TO ANTIGENIC ACTION 437 

phagocytosis of twenty-four-hour cultures by normal blood, the 
anticarbohydrate antibody tends to predominate as the pneumo- 
cocci approach the state in which they exist in the animal body. 
Enders and Wu 362 later reported (1934) that the opsonic titer of 
normal human serum could be practically eliminated by the addi- 
tion of the A carbohydrate, that is, the acetylated capsular poly- 
saccharide of Pneumococcus. 

Naturally Induced Immunity 

SPECIFIC ANTIBODIES IN THE BLOOD DURING PNEUMONIA 

Pneumococcus, upon entering the animal body and inciting mor- 
bid processes in the organs and tissues, by virtue of its several 
components arouses or stimulates physiological functions latent 
or active leading to immunity, and the products of the freshly ac- 
tivated functions may be detected and measured by the various 
serological reactions. The first intimation that immune substances 
arise in man as a result of pneumococcal infection came from the 
work of the Klemperers, 725 who discovered that serum taken from 
pneumonia patients after crisis displayed curative properties for 
experimentally infected rabbits. Similar protective antibodies were 
observed in the serum of pneumonia patients by Romer, 1155 in some 
cases several days after the onset of the disease. Then Neufeld and 
Haendel 990 succeeded repeatedly in demonstrating not only that 
there was a decided increase in protective substances for the mouse 
in the serum of pneumonia convalescents soon after crisis, but that 
the action was specific for heterologous as well as for homologous 
strains of pneumococci. The authors, therefore, were convinced 
that the crisis in lobar pneumonia depended upon the formation 
and specific action of antibodies. Seligman (1911 ), 1253 on the con- 
trary, found no difference in the degree of protective action of se- 
rum taken before or after the critical period. Strouse (1911), 1346 
using the Neufeld technique, was unable to detect opsonins in 
heated post-critical serum. However, after sensitizing the pneumo- 



438 BIOLOGY OF PNEUMOCOCCUS 

cocci with the serum, as recommended by Lamar, Strouse experi- 
enced no difficulty in obtaining phagocytosis. 

Eggers (1912), 349 by the plate method of determining bacteri- 
cidal action, found increased antipneumococcal properties devel- 
oping in the serum of pneumonia patients at or shortly after crisis 
and lasting for variable periods thereafter. Cases in which the 
apparently characteristic increase of bactericidal power did not 
occur presented irregularities either in course or in termination. 
Dochez (1912) 315 was successful in proving the presence of sub- 
stances protective for mice in the serum of patients ill with lobar 
pneumonia. All but one of the serums tested showed the ability to 
protect, the reactive substance appearing in only a few instances 
before the time of crisis, while in some other cases the protective 
power appeared to persist as long as the patient was under obser- 
vation. In some patients, protective antibodies either became evi- 
dent at some time after crisis or could not be demonstrated at any 
period of the disease. Dochez concluded that the appearance of 
specific protective substances in the serum of patients ill with 
lobar pneumonia suggested that these bodies might play a part in 
the mechanism of recovery. 

In the next year, Clough 240 reported the demonstration of 
protective antibodies, specific for the infecting strain of Pneumo- 
coccus, in the serum of the majority of patients after crisis or ly- 
sis. Phagocytic activity of the serum ran closely parallel with pro- 
tective power for mice. In addition to protective antibodies and 
opsonins, Lacy and Hartmann (1918) 770 reported that specific 
agglutinins usually appeared in the serum of pneumonia patients 
during or shortly after defervescence. In a second report, Clough 
(1919) 242 stated that the serum of 85 per cent of the patients ill 
with acute lobar pneumonia whom he had studied showed positive 
phagocytic activity after crisis or lysis, and that the serum of 79 
per cent of the cases showed agglutinative activity. In a few in- 
stances, positive results were obtained twenty-four hours or less 



HOST RESPONSE TO ANTIGENIC ACTION 439 

* 

before crisis. While phagocytic and agglutinative activity were 
observed for all the pneumococcal types tested, the reaction was 
always strictly limited to organisms homologous with those with 
which the patient was infected. 

Miiller (1923) 939 was inclined to ascribe to humoral antibodies 
only a minor part in recovery from pneumonia. His belief was 
based on negative results in tests for bactericidal power of serum 
from the majority of patients studied, and also on the fact that 
the author failed to observe any increase in the property during 
the course of the disease. Adler (1923), 3 on the contrary, reported 
that the serum of pneumonia patients developed the highest con- 
tent of bacteriotropic substances at the time of crisis. However, 
Baldwin and Rhoades (1925) 68 contended that recovery in pneu- 
monia is associated with the appearance of specific antibodies in 
the blood, and that the antagonistic action of the protective sub- 
stance is revealed by the fact that pneumococci and protective an- 
tibody rarely appear simultaneously in the circulating blood. The 
presence of protective substance in the blood practically always 
precluded a concurrent bacteriemia, but it did not in every case 
prevent toxemia, relapse, or the development of complications. 
Nevertheless, protective activity of the patient's serum appeared 
to be an important factor in overcoming pneumococcal infection. 

With refined technique for determining the antagonistic action 
of serum from pneumonia patients, Sia, Robertson, Woo, and 
Cheer (1925) 1274 found that in all cases of pneumonia studied the 
serum at or soon after crisis possessed the power to inhibit growth 
of pneumococci in rabbit serum-leucocyte mixtures. Before crisis, 
serum either lacked the property or exhibited it only to a slight 
degree. The titer reached its highest point three or four days fol- 
lowing crisis and then gradually diminished, although in one pa- 
tient recovering spontaneously from pneumonia due to Type I 
Pneumococcus antipneumococcic substances were still demonstra- 
ble in the blood for seventy days. In cases terminating fatally no 



440 BIOLOGY OF PNEUMOCOCCUS 

such substances could be found at any time during the course of 
the disease. In a second paper, Sia with Robertson and Woo 1273 
reported that the same conditions prevailed in pneumonia caused 
by pneumococci of Types I and II and Group IV. At the critical 
period, opsonins and agglutinins were also demonstrable in the 
blood. 

Other evidence of the production of an altered condition related 
to the immune state developing in the body of pneumonia patients, 
was the abolition of reactivity of the skin to pneumococcal fil- 
trates and extracts, as described by Herrold and Traut (1927). 637 
The serum of patients failing to react to the skin test partly or 
completely neutralized in vitro the antigenic substance in the ac- 
tive extracts and conferred protection upon mice. 

The time factor in the appearance of protective substances and 
agglutinins in the course of lobar pneumonia was investigated by 
Lord and Nesche (1929). 828 The authors were unable to find these 
antibodies in the blood of patients before a fall in temperature by 
crisis or lysis, but could demonstrate their appearance and con- 
tinued presence as soon as defervescence took place. Lord and 
Nesche attributed importance to the action of protective anti- 
bodies in bringing about recovery, since a large proportion of 
pneumonia patients with these substances in the blood conquered 
the disease, while the majority of patients without protective anti- 
bodies in their serum died. However, that immune bodies may be 
present in the early stages of untreated pneumonia is indicated by 
the experiments of Ward, 1480 who observed that the phagocytic 
titer of whole human blood was comparatively high against the in- 
fecting organism. For Ward, the fact pointed to a local rather 
than a general lowering of resistance in infection with Pneumo- 
coccus. 

In 1931, Lord and Persons, 838 continuing the study of the pro- 
duction of specific antibodies during pneumococcal pneumonia, re- 
ported that although in general the appearance of protective 
substance coincided sharply with the fall in temperature of the pa- 



HOST RESPONSE TO ANTIGENIC ACTION 441 

tient, the antibody might appear spontaneously in the blood serum 
as early as the third or fourth day of the disease and crisis and 
recovery might be delayed until the sixth to the tenth day. How- 
ever, recovery might occur without demonstrable protective sub- 
stance in the blood of patients whose serum later developed protec- 
tive properties. According to Lord and Persons, the amount of 
antibody appearing in the course of pneumococcal pneumonia was 
small and it might be present in the blood concurrently with sep- 
ticemia. The formation of protective substances by the patient 
gave no assurance that the infection would not progress to a fatal 
termination. 

In empyema fluids of pneumococcal origin, Floyd (1920) 456 
demonstrated the presence of specific precipitin and of much 
smaller amounts of agglutinin, and considered that their occur- 
rence was generally a favorable prognostic sign. Later, Finland 441 
reported (1932) that sterile pleuritic exudates from patients with 
lobar pneumonia contained actively acquired antibodies similar to 
those developing in the blood serum. 

A property of the serum of pneumonia patients which had es- 
caped previous notice was the capacity to precipitate in high titer 
the non-protein somatic substance — the C Fraction — derived from 
pneumococci. Tillett and Francis (1930) 1409 tested serum obtained 
from patients during illness and convalescence for antibodies spe- 
cifically reactive with this chemically distinct carbohydrate of 
Pneumococcus. The results, when correlated with the course of the 
disease, demonstrated differences in the occurrence of each qualita- 
tively distinct antibody. Strangely enough, the precipitating ac- 
tion of the serum on the somatic carbohydrate developed in the 
very early stages of the disease, only to disappear at the time of 
crisis. Precipitation of Fraction C was not limited to the serum 
of individuals ill with pneumococcal infection and, in the few cases 
available for comparative tests, definite reactions were obtained 
only in streptococcal and staphylococcal infections and in acute 
rheumatic fever. 



442 BIOLOGY OF PNEUMOCOCCUS 

IMMUNE SUBSTANCES IN SPUTUM 

Lord and Nye (1921) 831 " 3 observed that purulent sputum col- 
lected during life and the pulmonary exudate obtained at necropsy 
from the later stages of lobar pneumonia commonly eroded the 
surface of Loeffler's blood serum and, in a separate communication, 
Nye 1021 reported that washed cellular suspensions of pneumonic 
lungs, previously preserved with chloroform and toluene, contained 
a proteolytic ferment, derived chiefly from the leucocytes of the 
exudate. Eddy (1928) 348 found that filtrates from sputum ob- 
tained after crisis from patients with lobar pneumonia conferred a 
certain degree of protection on mice, with sometimes only a delay 
in the time of death. The effect was never produced by filtrates of 
sputum obtained before crisis or from fatal cases, nor was the fil- 
trate active with organisms other than those of the type infecting 
the patient. The sputum of two patients displayed proteolytic ac- 
tion, but Eddy was unable to demonstrate bacteriophage in any of 
the specimens of sputum. The observation recalls that of Dick 
(1912), 314 who by noting the optical rotation of mixtures of serum 
from pneumonia patients taken at the time of crisis found that a 
decrease in optical rotation occurred at that time and not before 
or after the critical period. Dick ascribed the phenomenon to pro- 
teolytic activity of the serum. 

ANTAGONISTIC SUBSTANCES IN PNEUMOCOCCAL EXUDATES 

Proceeding from the conception that increased acidity in pneu- 
mococcal cultures might find an analogy in the pneumonic lung, 
Lord (1919), 826 upon testing the hydrogen ion concentration of 
morbid exudates, found higher acidity in three of four cases than 
that in the press juice of the unaffected lung. Lord suggested that 
increase in acidity in the diseased pulmonary tissue might favor 
enzymatic action as well as inhibit pneumococcal growth. 

In further studies on pneumonic exudates, Lord with Nye 
(1921 ), 8301 demonstrated the presence of a proteolytic enzyme. 
The enzyme remained active after eighteen months' preservation 



HOST RESPONSE TO ANTIGENIC ACTION 443 

and resisted heating at 65° for one hour, but was destroyed after 
heating for the same period at 75°. No dialysis of the enzyme 
could be demonstrated. The activity of the enzyme persisted in 
concentrations of sodium chloride varying from normal to thirty- 
two times normal. In exudates, antigenic substances were also 
found, evidently arising from the dissolution of pneumococci in the 
infected lung. The presence of specific precipitinogen was disclosed 
when the exudate was mixed with homologous antipneumococcic se- 
rum. After testing extracts of affected lungs, Lord and Nye 833 fur- 
ther concluded that the pneumonic lung contained a soluble sub- 
stance inhibiting agglutination of fixed types of pneumococci by 
homologous serum. An analogous substance was found by Ward 
(1932) 1483 in the filtrate of a lung obtained at necropsy from a pa- 
tient dying from pneumonia due to Type III Pneumococcus. The 
substance was similar to that present in somewhat lower concen- 
tration in broth cultures of Type III Pneumococcus which dis- 
played powerful antibactericidal action. 

Artificially Induced Immunity 

The antigenic properties of Pneumococcus and the manifesta- 
tions of the immunological reaction of the animal body to anti- 
genic stimuli have already been treated in the text in some detail. 
However, in order that the presentation of the features of the ani- 
mal hosts' response may be orderly and complete, a brief synopsis 
is made at this point of the factors that are chiefly concerned in 
the artificial production of the immune state. 

ACTIVE IMMUNITY 

The ability of pneumococcal materials, purposely introduced, to 
raise the resistance of susceptible animals to infection with Pneu- 
mococcus, with the accompanying elaboration of demonstrable 
specific immune substances or antibodies, is dependent upon the 
nature of the material employed and also upon the special racial 
and individual peculiarities of the animal treated. The more 



444 BIOLOGY OF PNEUMOCOCCUS 

closely the state of the antigen approaches that of the vigorous, 
living, virulent cell, the greater the specificity and the complete- 
ness, within certain limits, of the immunity and of the antibodies 
evoked. The robust organism with its protein and carbohydrate 
constituents in fully developed and unaltered condition exerts the 
greatest antigenic action. The cell should be living, or devitalized 
at the peak of its anabolic activities by heat or by such chemical 
agents as rob the bacterium of the ability to propagate without 
disturbing its chemical integrity. The greater the mass, within 
certain limiting zones, the more energetic the specific antigenic 
stimulation; while the proper spacing of injections and the route 
by which the antigen enters the body may affect the specificity and 
the quantity of antibodies produced. 

The protein of the pneumococcal cell is antigenic only in the 
sense that its administration by parenteral routes results in the 
appearance of humoral antibodies specific for the bacterial spe- 
cies and not for the serological type of the organism injected. The 
capsular polysaccharide, besides orienting the antigenic action 
of its conjugated protein when its molecular configuration is un- 
disturbed, is antigenic in itself, and it is this fraction of the pneu- 
mococcal cell that determines the type-specificity of the immune 
response. The somatic carbohydrate — the C Fraction — if it has 
any antigenic action, apparently has a subordinate and as yet 
unknown share in the immunizing action of Pneumococcus. 

In an animal of a susceptible species, unaffected by any debili- 
tating condition, the parenteral introduction and, to a much less 
degree, the oral administration of properly chosen pneumococcal 
antigens, decreases susceptibility to pneumococcal infection and 
arouses physiological functions latent in the cells of the body. 
These functions result in the extrusion into the circulation of sub- 
stances corresponding in type to the kind of antigen administered, 
and demonstrable and measurable by the various serological reac- 
tions. In addition to the appearance of humoral antibodies, the 
somatic cells undergo changes in reactivity to pneumococcal ma- 



HOST RESPONSE TO ANTIGENIC ACTION 445 

terials. Of the immune substances engendered, the agglutinins ag- 
glomerate the cocci and facilitate their removal from the circu- 
lating blood and the precipitins combine with the polysaccharide 
present in the capsule or released in the disintegration of the bac- 
terial cell, while the opsonins render the organisms susceptible to 
the phagocytic action of the leucocytes and of certain of the fixed 
cells of the body. 

The immunity thus established by artificial means may become 
apparent within the space of a few days after the administration 
of antigen and may persist for longer or shorter periods depend- 
ing upon the total amount of antigenic material injected, the 
spacing and repetition of the injections, and the ability of the 
tissues to continue their special functions. Immunity to Pneumo- 
coccus is, at best, a transient condition, and unless the tissues are 
fortified by continued specific stimulation, the antibodies thus ar- 
tificially induced shortly disappear from the blood and the animal 
again becomes vulnerable to the pathogenic action of Pneumo- 
coccus. 

PASSIVE IMMUNITY 

The blood or serum of an immune animal, whether transferred 
through the placental circulation or artificially injected into the 
body of a susceptible animal, carries its complement of antibodies 
that serve to convert susceptibility into resistance; the degree of 
resistance depends upon the potency of the serum in immune sub- 
stances, the volume of serum, the route and frequency of adminis- 
tration, and the capacity of the recipient to utilize the antibodies 
so conferred. In the passively immunized animal, the type of im- 
munity acquired corresponds in specificity to the immunity of the 
donor. 

References to the inheritance of immunity to pneumococcal in- 
fection are singularly rare. By inference, it might be assumed 
from the work of Irwin and Hughes 670 that native resistance may 
be increased by selective breeding, but in that case the ability of 



446 BIOLOGY OF PNEUMOCOCCUS 

the animals to withstand infection is due to transmitted constitu- 
tional factors unrelated to those which account for the specific 
immune state. The report of Eguchi (1925) 351 is one of the few 
communications dealing with this phase of immunity. The young 
of female mice which, during the period of gestation and lacta- 
tion, had received repeated intravenous injections of killed Type 
I pneumococci were found to be immune to organisms of the same 
type as those employed for immunizing the mother. The ability of 
the mother to protect the offspring disappeared between the six- 
teenth and twenty-eighth day after the last immunizing injection. 
Eguchi believed that protection was conferred through the milk of 
the immune mother. The results of attempts to transmit specific 
immunity to the progeny by similarly treating the male parent 
were inconclusive. 

Another natural medium for the transference of specific immune 
bodies is the serum of patients convalescent from pneumococcal in- 
fection. Gundel (1931) 565 studied the curative action of conva- 
lescent serum and while it was found to be inferior to the usual 
therapeutic serums of animal origin, yet, according to Gundel, 
specific therapy with human serum possesses advantages over the 
use of animal serum in the avoidance of anaphylaxis and serum 
disease. Other phases of passive immunity will be described in those 
sections of the text where a discussion of the mechanism is espe- 
cially pertinent to the particular subject under consideration. 

Allergy and Anaphylaxis 

In addition to rendering a susceptible animal immune to infec- 
tion by Pneumococcus, the intact pneumococcal cell and its sepa- 
rate or conjugated components possess the capacity to alter the 
reactivity of the body tissues to derivatives of the coccus when 
parenterally introduced. Animals so sensitized may develop acute 
fatal anaphylactic shock when appropriately injected with cer- 
tain of the derivatives of Pneumococcus, while the uterus and the 



HOST RESPONSE TO ANTIGENIC ACTION 447 

dermal tissues of animals thus treated display a newly established 
function when brought into contact with these products. Actively 
acquired hypersensitivity may be duplicated by the injection of 
normal animals with some forms of antipneumococcic serum. 

The interaction between specific immune serum and the pneumo- 
coccal cell resulting in the formation of a substance capable of 
eliciting acute anaphylactic shock in the guinea pig was first 
demonstrated by Neufeld and Dold (1911 ). 981 Cocci sensitized 
with homologous antiserum and injected into guinea pigs regu- 
larly caused the acute death of the animals. The effect was as- 
cribed by Neufeld and Dold to the production of anaphylatoxin. 

ACTIVELY INDUCED SENSITIZATION 

In 1911, Rosenow 1165 claimed that, by the subcutaneous, intra- 
venous, intrapleural, and intraperitoneal injection of killed pneu- 
mococci or of filtered pneumococcal extracts into guinea pigs, he 
could so sensitize the animals that they responded with severe in- 
toxication when the same bacterial extract was injected eight to 
twelve days later into a vein or into the heart. In a subsequent 
study, Rosenow (1912) 1166 was able to obtain highly toxic sub- 
stances from Pneumococcus through autolysis, from Pneumococ- 
cus-leucocyte mixtures, and by the action of normal and of im- 
mune serum on the organisms. These toxic substances evoked in 
normal guinea pigs symptoms indistinguishable from the symp- 
toms of immediate anaphylaxis. The action of the poisonous sub- 
stances as well as of the preparations of Cole, Weiss, and others 
has been discussed in Chapter III, and undoubtedly is to be at- 
tributed to histamine-like protein-degradation products. 

Clough (1915) 241 employed saline extracts of washed, dried, and 
ground pneumococci as sensitizing antigens and as the intoxicat- 
ing agent. The extracts so prepared were sufficiently toxic to cause 
anaphylactoid reactions in normal guinea pigs. However, by pre- 
cipitating the extracts with alcohol, Clough isolated a substance, 



448 BIOLOGY OF PNEUMOCOCCUS 

assumed to be protein, that was antigenic in the sense that it could 
sensitize normal guinea pigs and specifically intoxicate animals so 
treated. 

The allergic state produced in guinea pigs by the intraperi- 
toneal injection of killed broth cultures of Type I pneumococci 
was observed by Mackenzie (1925). 844 During the course of arti- 
ficially induced, active immunity, the animals at times showed ana- 
phylactic symptoms after the injection of the immunizing anti- 
gens. Bull and McKee (1929), 180 in an investigation of sensitiza- 
tion of the rabbit resulting from acute experimental infection with 
Type I Pneumococcus, found that the animals had acquired both 
dermal and systemic hypersensitiveness to pneumococcal autoly- 
sate. Sensitivity appeared within forty-eight hours after infec- 
tion and persisted for at least four months, apparently reaching 
its height shortly after recovery from infection. Rabbits im- 
munized by the injection of killed and living cultures also became 
hypersensitive to autolysates but not to so high a degree as did 
the animals recovering from infection. 

Although Avery and Tillett (1929) 60 were unable to sensitize 
guinea pigs with the type-specific carbohydrates of pneumococci 
of Types I, II, and III, Tillett, Avery, and Goebel, 1407 in the same 
year, demonstrated the essential function of the carbohydrate 
fraction in determining the specific sensitizing and anaphylactic 
action of the carbohydrate-protein complex. Artificially prepared 
gluco-globulin and galacto-globulin possessed the property of ac- 
tively sensitizing guinea pigs so that the guinea pigs, when in- 
jected twenty-one days later with sugar-proteins containing car- 
bohydrate identical with that present in the sensitizing antigen 
regardless of the kind of protein with which it was combined, were 
subject to acute anaphylactic shock. Moreover, the unconjugated 
glucosides, although themselves incapable of inducing shock, in- 
hibited the anaphylactic reaction when injected immediately prior 
to the introduction of the toxigenic sugar-protein into the spe- 
cifically sensitized guinea pig. The protective, or anti-anaphylac- 



HOST RESPONSE TO ANTIGENIC ACTION 449 

tic action of the glucoside disappeared within two hours after in- 
jection and, in order to elicit the phenomenon, the carbohydrate 
had to be the same as that combined in the sugar-protein complex. 

PASSIVE SENSITIZATION 

From the collective experience of immunologists it becomes evi- 
dent that some of the isolated constituents of the pneumococcal 
cell are incapable of so sensitizing the guinea pig as to render the 
animal susceptible to anaphylactic intoxication upon the paren- 
teral introduction of Pneumococcus or its derivatives. However, 
that an animal may be rendered highly sensitive to pneumococcal 
substances by the injection of specific antipneumococcic serum is 
supported by ample evidence. Weil and Torrey 1508 passively sensi- 
tized guinea pigs by the injection of serum from pneumonia pa- 
tients. When autolysates of pneumococci were added to the uterus 
of the treated animal, positive contractions occurred only when 
the serum employed originated from a case of pneumococcal pneu- 
monia. By means of the same method, Zinsser and Mallory 1582 ob- 
tained positive reactions with the uteri of guinea pigs passively 
sensitized with antipneumococcic serum. More conclusive were the 
observations of Tomcsik (192T), 1415 who demonstrated the ability 
of the soluble specific substance of Pneumococcus to produce ana- 
phylactic shock in passively sensitized guinea pigs. The purified 
capsular polysaccharide of Pneumococcus, though apparently de- 
void of sensitizing action, can induce rapid and fatal anaphylactic 
shock when injected intravenously into guinea pigs passively sensi- 
tized with the precipitating serum of rabbits actively immunized 
with pneumococci of the homologous type, the reactions induced 
being type-specific. Avery and Tillett, 60 who reported the observa- 
tion, also drew attention to the fact that there was a complete ab- 
sence of anaphylactic response to pneumococcal carbohydrate in 
guinea pigs similarly treated with antipneumococcic horse serum. 

As in their experiments on the production of active specific 
sensitization with artificially prepared sugar-proteins, Tillett, 



450 BIOLOGY OF PNEUMOCOCCUS 

Avery, and Goebel 1407 demonstrated further the dependence of the 
specificity of the anaphylactic phenomenon upon the carbohydrate 
portion of these compounds. Guinea pigs passively sensitized with 
the serum of rabbits immunized with an artificial gluco-globulin 
exhibited typical anaphylactic shock when subsequently injected 
with gluco-albumin ; the serum of rabbits immunized with another 
sugar-protein, galacto-globulin, similarly sensitized guinea pigs 
to galacto-albumin. The reactions, in each instance, were specific 
and depended for their specificity upon the carbohydrate com- 
ponent and not on the protein fraction of the synthesized sugar- 
protein. The authors found that anaphylactic shock could be in- 
duced by uncombined globulin in guinea pigs passively sensitized 
with either antigluco-globulin serum or antigalacto-globulin se- 
rum, and that the globulin was similarly effective in animals ac- 
tively sensitized with gluco-globulin or galacto-globulin. However, 
the reactions provoked by globulin alone were dependent upon the 
common protein present in the antigens and exhibited only species 
specificity. 

The haptenic participation of the A carbohydrate in the ana- 
phylactic reaction was demonstrated by Enders (1930). 358 Guinea 
pigs injected intraperitoneally with one to two cubic centimeters 
of the anti-A rabbit serums used in the precipitin tests failed to 
develop symptoms of anaphylaxis upon intravenous injection of 
varying quantities of the purified specific carbohydrate. Nor was 
the antibody against the nucleoprotein or rough autolysate pres- 
ent in sufficient concentration in these serums to confer on guinea 
pigs anaphylactic sensitivity to the substances. Again, no ana- 
phylaxis developed in animals treated with the serum when autoly- 
sates derived from virulent strains of either Type II or Type III 
pneumococci were introduced intravenously. Type I anti-A serum, 
however, regularly conferred upon guinea pigs a very high degree 
of anaphylactic sensitivity to the autolysate derived from Type I 
Pneumococcus. The "normal" antipneumococcic Type I rabbit 
serum, from which the specific anticarbohydrate antibody had 



HOST RESPONSE TO ANTIGENIC ACTION 451 

been removed, also rendered guinea pigs anaphylactically hyper- 
sensitive to the A substance in the homologous autolysate. Before 
the removal of the anticarbohydrate antibody by precipitation in 
vitro, the serum sensitized guinea pigs to the type-specific soluble 
substance. After the antibody had been eliminated, animals in- 
jected with the serum failed to react to the injection of the car- 
bohydrate, but responded typically when injected with the autoly- 
sate from virulent organisms of Type I. Furthermore, the animals 
showed no symptoms following the intravenous administration of 
autolysate derived from a rough strain of Type I or of autoly- 
sates of smooth strains of Types II and III. 

The experiments in anaphylaxis showed that in the serum of 
rabbits receiving injections of formalinized pneumococci an anti- 
body develops that reacts specifically with an antigen found in the 
autolytic products of Type I Pneumococcus. Similar autolysates 
of the other two types (II and III) of this organism, as well as 
rough strains derived from homologous or heterologous types, 
elicited no anaphylactic symptoms in guinea pigs. Under the same 
conditions, the nucleoprotein was likewise incapable of provoking 
specific shock. That the antigen in Type I autolysate responsible 
for the anaphylactic symptoms was not the specific carbohydrate 
appeared to Enders to be shown not only by the failure of the lat- 
ter material, when pure, to produce shock in guinea pigs sensitized 
with anti-A rabbit serum, but also by the experiments with rabbit 
serum from which the carbohydrate antibody originally capable 
of sensitizing the animals had been removed, leaving unimpaired 
the power of the serum to sensitize the animals to the A substance. 

The cellular carbohydrate from Type I Pneumococcus, like- 
wise, caused immediate, lethal anaphylactic intoxication in guinea 
pigs previously injected with Type I antipneumococcic rabbit 
serum. Schiemann, Loewenthal, and Hackenthal 1231 reported a 
similar effect obtained with the carbohydrate preparation made 
by the method of Schiemann and Casper. 1228 These results, taken 
with further observations of Avery and Goebel (1931) 45 on the 



452 BIOLOGY OF PNEUMOCOCCUS 

immunological action of an antigen prepared by combining the 
capsular polysaccharide of Type III Pneumococcus with horse 
serum globulin, fully justify the conclusion that it is the soluble 
specific carbohydrate which determines the type-specificity of the 
hypersensitive state and of the anaphylactic reaction. 

The contrasting differences in the sensitizing properties of se- 
rum from the immune rabbit and the immune horse were studied by 
Mehlman and Seegal (1934). S89 In conformity with the results of 
Avery and Tillett, guinea pigs injected parenterally with anti- 
pneumococcic rabbit serum were thrown into anaphylactic shock 
by the intravenous injection twenty-four hours later of the capsu- 
lar carbohydrate of pneumococci corresponding in type to that of 
the immune serum. Guinea pigs similarly prepared with antipneu- 
mococcic horse serum were not susceptible to an otherwise shock- 
ing dose of the homologous antigen. Uteri removed from guinea 
pigs sensitized passively with the immune rabbit serum contracted 
characteristically on contact with the homologous specific carbo- 
hydrate, whereas uteri of guinea pigs injected with immune horse 
serum failed to react in this manner. Tests made to determine a 
possible anti-alexic activity of parenterally injected antipneumo- 
coccic rabbit and horse serum failed to show any marked differ- 
ence between the two serums. In a second communication, Mehl- 
man and Seegal 890 demonstrated in mice the same difference be- 
tween the sensitizing property of antipneumococcic serum from 
the rabbit and from the horse. The sensitizing property of immune 
rabbit serum appeared to be independent of protective action, 
since the serum of both the immune rabbit and horse were equally 
effective in curing mice of infection with Pneumococcus.* 

The comparative inability of antipneumococcic horse serum to 
sensitize the guinea pig passively to Pneumococcus is also evident 
when sensitivity is tested by intracutaneous injection of soluble 
specific substance. According to Mehlman and Seegal (1934), 

* Differences in the nature of antibodies in immune rabbit and horse serum 
have been discussed in Chapter XI. 



HOST RESPONSE TO ANTIGENIC ACTION 453 

preparations of Type I antipneumococcic serum, obtained from 
rabbits, sensitized the albino guinea pig so that an injection of 0.1 
cubic centimeter of a 1 to 10,000 dilution of Type I polysaccha- 
ride into the skin of the ear elicited a positive reaction. However, 
when antipneumococcic serum from the horse was employed, the 
same dose of antigen gave variable results, although the injection 
of twice the amount evoked positive reactions. Experiments on the 
distribution of immune horse serum as compared with that of rab- 
bit serum in the circulation and organs of passively immunized 
guinea pigs failed to disclose any explanation for the differences 
in the sensitizing action of the two serums. 

Brown 155 " 6 reported similar observations on the ability of anti- 
pneumococcic rabbit serum to sensitize passively the guinea pig to 
the specific carbohydrate of Type I Pneumococcus. Brown found 
that the minimal amount of antiserum necessary to sensitize the 
guinea pig so that fatal shock followed the injection into the 
heart of the soluble specific substance was always greater than 
the quantity of serum required to sensitize to the cellular carbo- 
hydrate of Wadsworth. However, the minimal amount of each of 
the specific carbohydrates which induced fatal shock in guinea 
pigs previously sensitized by the injection of one cubic centimeter 
of immune rabbit serum was approximately the same. 

The protective or inhibitory action of artificial, conjugated 
sugar-proteins on the anaphylactic reaction of guinea pigs pas- 
sively sensitized with antipneumococcic rabbit serum, as reported 
by Tillett, Avery, and Goebel, 1407 was confirmed by Brown 156 in the 
case of the soluble specific substance and of the cellular carbo- 
hydrate. A subcutaneous injection of a suitable dose of soluble 
specific substance of Type I Pneumococcus given to guinea pigs 
twenty-four hours after passive sensitization with Type I anti- 
pneumococcic rabbit serum completely desensitized the animals 
to intracardial injection twenty-four hours later of capsular poly- 
saccharide, but not to a similar injection of cellular carbohydrate. 
The latter substance, however, completely desensitized the guinea 



454 BIOLOGY OF PNEUMOCOCCUS 

pigs to the anaphylactic action of the soluble specific substance 
and brought about partial desensitization to the cellular carbo- 
hydrate. When capsular polysaccharide prepared according to the 
original method of Heidelberger and Avery (probably the deacety- 
lated carbohydrate) was added to the minimal dose of serum neces- 
sary to sensitize, and the mixture injected at once into guinea 
pigs, the animals failed to become hypersensitive to subsequent 
intracardial injection of the same preparation, but acquired sensi- 
tivity to the cellular carbohydrate. However, when cellular carbo- 
hydrate was substituted for soluble specific substances in the ex- 
periment, no sensitization to either capsular or cellular carbo- 
hydrate could be demonstrated. 

The results reported by Brown were in accord with observations 
previously described by Wadsworth and Brown (1933). 1468 Spe- 
cific immune serum, from which the precipitate, formed on the ad- 
dition of SSS, had been removed, was still capable of sensitizing 
guinea pigs to the cellular carbohydrate but not to soluble specific 
substance. Antiserum, after precipitation with cellular carbohy- 
drate, failed to sensitize the animals to either substance. The dif- 
ference in the action of the two preparations of specific pneumo- 
coccal polysaccharides was undoubtedly due to the lack of molecu- 
lar completeness of the preparation of soluble specific substance 
employed in the experiments. 

RELATION OF PNEUMOCOCCAL ALLERGY TO PNEUMOCOCCAL IM- 
MUNITY 

The experiments of Mackenzie 844 suggest a lack of correlation 
between the hypersensitive and the immune states in guinea pigs 
treated with Pneumococcus or its extracts. During the course of 
immunity produced by the intraperitoneal injection of killed and 
living broth cultures of virulent pneumococci, while the animals 
showed high resistance to infection and possessed strong protec- 
tive power in their serum, hypersensitiveness might or might not 
be present. Mackenzie concluded that anaphylaxis to pneumo- 



HOST RESPONSE TO ANTIGENIC ACTION 455 

coccal protein was merely a concomitant of immunity without hav- 
ing any significant part in the immunological mechanism. 

Sharp and Blake (1930) 1260 differed with the conclusions of 
Mackenzie and maintained that in rabbits a fairly close parallel- 
ism exists between cutaneous and pulmonary hypersensitiveness to 
pneumococcal autolysate, and that the inflammatory response of 
pulmonary tissue, resulting from contact with autolysate, depends 
on the allergic state of the animal rather than on inherently in- 
jurious substances in the autolysate. Sharp and Blake interpreted 
their observations as being in harmony with the theory that al- 
lergy may play a part in the pathogenesis of pneumococcal pneu- 
monia in man. 

Julianelle and Rhoades (1932), 696 in a study of the reaction of 
the lungs of rabbits to infection caused by intravenous injections 
of Pneumococcus, found that reactions occurred irregularly in the 
lung, and that in the lungs in which reactions did occur, the his- 
tological changes were no different in normal rabbits from the 
changes in rabbits made resistant by previous intravenous or in- 
tracutaneous injections of pneumococci. The authors concluded 
that the experiments afforded no evidence to support the view that 
the lesions in the lungs of rabbits, following the intravenous injec- 
tion of pneumococci, were modified by any previous state of sensi- 
tivity. 

Dermal Allergy 

SKIN REACTIONS IN EXPERIMENTAL. ANIMALS 

The development of an allergic condition of the skin in guinea 
pigs following the intracutaneous injection of an alkaline extract 
of pneumococci occurred in experiments described by Mackenzie 
and Woo (1925). 845 About two-thirds of the animals so treated 
acquired cutaneous hypersensitivity but, upon the continuance of 
the injections, the capacity of the dermal tissues to react ceased. 
Neither the animals manifesting skin allergy nor those that failed 
to develop this type of hypersensitiveness showed any significant 



456 BIOLOGY OF PNEUMOCOCCUS 

alteration in susceptibility to pneumococcal infection by intra- 
peritoneal inoculation. Similarly, animals desensitized by repeated 
intracutaneous injection after the appearance of allergy ex- 
hibited an unaltered susceptibility to infection. 

In 1927, Zinsser and Grinnell 1581 investigated the action in the 
skin of normal guinea pigs of various bacterial autolysates in- 
cluding those prepared from pneumococci. Reactions were noted 
only in the case of well-grown animals, whereas young guinea pigs 
almost invariably gave a negative reaction on the first test. On 
further study it was found that the cutaneous reactions were 
manifestations of hypersensitiveness which could be elicited by 
bile solutions of Pneumococcus as well as by similar autolysates. 
Sensitization could be induced by the previous injection of dead, 
intact bacteria, as well as of autolysates, but the actual skin re- 
action was elicited only by autolysates. When properly treated, 
insensitive animals could be rendered sensitive within a space of 
five to seven days by the daily injection of autolysate. The sensi- 
tivity thus produced was related to the bacterial species and not 
to pneumococcal type. Using the rabbit, Julianelle (1930), 689 
after subjecting the animals to repeated intracutaneous injections 
of heat-killed pneumococci, demonstrated increased skin activity 
which reached a maximum after four to six injections had been 
given and then diminished. After regression of the reaction to the 
first injection of antigen into the skin, there frequently followed 
a recrudescence or exacerbation of the reaction. By injecting the 
serum of a highly reactive animal into a normal rabbit, the author 
was unable to confer the property of skin-reactivity upon the re- 
cipient. 

In another paper, Julianelle 690 communicated further results ob- 
tained in rabbits during a study of actively induced skin-sensi- 
tivity to derivatives of Pneumococcus. Positive reactions were 
evoked in animals that had previously received repeated intra- 
cutaneous injections of heat-killed pneumococci, of the pneumo- 
coccal nucleoprotein, and of a solution of the bacterial cell from 



HOST RESPONSE TO ANTIGENIC ACTION 457 

which the acid-precipitable and heat-coagulable proteins had been 
removed. The reaction appeared to be species-specific but not 
type-specific. A similar skin reaction to the protein of Pneumo- 
coccus occurred in rabbits following the repeated intracutaneous 
or intravenous administration of heat-killed organisms or their 
protein derivatives. The reactive function, although evidently re- 
lated to the presence of humoral species-specific antibodies, oc- 
curred independently of resistance to infection. 

A phenomenon related to skin sensitivity was the increased re- 
active power of the eye of rabbits immunized with heat-killed sus- 
pensions of smooth and rough pneumococci separately, as de- 
scribed by Julianelle. 691 Positive reactions could be elicited in im- 
mune animals by the instillation of nucleoprotein, or of a solution 
from which the acid-precipitable and heat-coagulable proteins had 
been removed. No reaction followed the similar application of liv- 
ing, rough cells or the protein-free, type-specific polysaccharide of 
Pneumococcus. Rabbits receiving intravenous injections of the in- 
tact cell or of the soluble derivatives of Pneumococcus failed to 
develop eye-reactivity, but acquired the property as a result of ex- 
perimental infection with Pneumococcus. From the collected ex- 
perimental evidence, Julianelle 692 concluded that the injection of 
heat-killed pneumococci into the skin created a special kind of 
skin and eye sensitiveness unrelated to the presence of circulating 
antibodies and not transferable from sensitive to normal rabbits. 

In 1932, Julianelle with Morris, 693 by means of cutaneous and 
ophthalmic tests, disclosed another indication of a basic relation 
between pneumococci and streptococci and, in addition, a differ- 
ence between the specificity of the reaction of the eye and of the 
skin of sensitized rabbits. Following repeated intracutaneous in- 
jections of heat-killed smooth and rough forms of Pneumococcus, 
rabbits acquired increased dermal sensitivity to both pneumococci 
and streptococci, while the injection of heat-killed indifferent 
streptococci produced skin sensitivity to both species of organ- 
isms. Injections of pneumococci into the skin were followed in 



458 BIOLOGY OF PNEUMOCOCCUS 

some rabbits by eye sensitivity to pneumococcal protein but not to 
streptococcal protein or to suspensions of either living organism, 
whereas similar injections of indifferent streptococci were fol- 
lowed, in some rabbits, by eye sensitivity to suspensions of living 
indifferent streptococci but not to suspensions of rough pneumo- 
cocci or to nucleoprotein of either organism. Measured in terms 
of bacterial specificity, therefore, eye sensitivity appeared to pos- 
sess a degree of specificity not shared by skin sensitivity. A col- 
lateral observation of Julianelle and Morris was to the effect that 
the serum of rabbits injected intracutaneously with pneumococci 
or indifferent streptococci contained agglutinins for both rough 
pneumococci and indifferent streptococci, but only those rabbits 
which received injections of Pneumococcus acquired resistance to 
pneumococcal infection, 

Barnes* studied cutaneous reactions to pneumococcal protein 
and capsular polysaccharide in rabbits both actively and pas- 
sively immunized against Pneumococcus and in horses undergoing 
active immunizing treatment for the production of antipneumo- 
coccic serum. In rabbits actively immunized with Type II vaccine, 
positive skin reactions were obtained following the intradermal in- 
jection of the homologous carbohydrate. The reaction consisted 
of an area of swelling and erythema having an average diameter 
of fifteen to twenty millimeters or more, and the crest of the swol- 
len area was raised to a height of four to five millimeters. Positive 
reactions usually appeared within two hours after the injection 
as edematous areas which, within eighteen to twenty-four hours, 
became indurated and then persisted for more than two weeks. 
Similar injections into normal rabbits or rabbits immunized with 
heterologous strains of pneumococci were never followed by this 
type of reaction, although occasionally an early, transient ery- 
thema and slight swelling would appear, only to disappear within 
eighteen hours. Intradermal injections of somatic protein, in the 
amounts used, prepared from Type III pneumococci failed to 

* Unpublished observations. 



HOST RESPONSE TO ANTIGENIC ACTION 459 

elicit reactions of significance either in normal rabbits or in rab- 
bits previously immunized with killed pneumococci of the same 
type. 

When a mixture of equal parts of 1 to 1,000 Type II polysac- 
charide and undiluted homologous immune rabbit serum was in- 
jected intradermally into normal rabbits, there developed a cu- 
taneous reaction similar to that observed in actively immunized 
rabbits when similarly injected with the carbohydrate alone. In 
the few animals tested, the reaction following the antigen-antibody 
mixture was not so severe or so lasting as that evoked by the in- 
jection of polysaccharide into the skin of an actively immunized 
rabbit. 

In another experiment the precipitin titer was determined on a 
sample of serum from a rabbit which had been actively immunized 
with Type II pneumococci and which exhibited a typical cuta- 
neous reaction to the corresponding polysaccharide. Then, on the 
basis of body weight, a normal rabbit was given an intravenous in- 
jection of concentrated Type II antipneumococcic horse serum in 
an amount calculated to establish in the serum of the rabbit so 
treated a precipitin titer corresponding to that of the serum of 
the actively immunized animal. A precipitin test on serum from a 
test bleeding, taken one hour after the injection of immune serum, 
indicated that the desired result had been obtained. Skin tests with 
Type II polysaccharide performed at one-half-hour, one-hour, and 
two-hour intervals after the serum injection yielded reactions ap- 
parently identical with those observed when the same antigen was 
injected intradermally into rabbits actively immunized with Type 
II pneumococci. A repetition of the skin tests showed that positive 
reactions could be elicited for at least four days after the injec- 
tion of serum. It was found, however, when normal horse serum 
was substituted for the specific, concentrated, immune horse se- 
rum, that no reaction took place. 

In horses during the course of active immunization, positive 
cutaneous reactions could be obtained by the intradermal injec- 



460 BIOLOGY OF PNEUMOCOCCUS 

tion of homologous capsular polysaccharide. The reaction tended 
to be larger in area but not so sharply defined as that in the ac- 
tively immune rabbit. A peculiar phenomenon observed was the 
disappearance and subsequent recurrence of the reaction in some 
instances without intervening intradermal injections.* The long- 
est period of time over which spontaneously recurring reactions 
were observed was slightly more than six months. The injection of 
pneumococcal protein into immune horses, and of type-specific 
polysaccharide or protein into normal horses or into horses im- 
munized with heterologous types of pneumococci, resulted in no 
reaction. The experiments, though incomplete, point to a local 
reaction in the dermal tissues between antigen and circulating 
antibody or to a fixation of antigen by antibody in the dermal 
cells rather than to a hypersensitive state of the cutaneous tissues. 
Francis and Tillett (1931) 479 succeeded in demonstrating skin 
reactions to capsular polysaccharides of pneumococci of Types I, 
I, and III in rabbits actively or, under certain conditions, pas- 
sively immunized to organisms of one or another of these types. 
The reaction resembled that of Arthus, and was demonstrable only 
when type-specific precipitins for the homologous polysaccharide 
were present in the blood of the rabbit. A reaction was elicited in 
84 per cent of actively immunized animals, the serum of which 
contained type-specific precipitins, and a positive result was ob- 
tained in all rabbits passively immunized with antipneumococcic 
horse serum. Attempts to transfer the reactive principle from im- 
mune rabbit to normal rabbit were unsuccessful. The recipients 
in the latter group possessed no demonstrable circulating type- 
specific precipitins. Francis and Tillett concluded that the reac- 
tion produced by specific capsular carbohydrates is always asso- 
ciated with a well-grounded type-specific immunity. 

SKIN REACTIONS IN LOBAR PNEUMONIA 

The value of skin reactions to tuberculin and to diphtheria 
* Compare with observation of Julianelle, page 456, ante. 



HOST RESPONSE TO ANTIGENIC ACTION 461 

toxin in determining susceptibility or resistance to infection stimu- 
lated interest in a similar application of pneumococcal antigens. 
Weil (1916) 1507 employed a saline autolysate of Pneumococcus 
(usually of Type I) and injected the material intracutaneously in 
the scapular region. The injection was immediately followed at 
times in pneumonia patients, and in normal subjects as well, by a 
superficial, ill-defined, cutaneous blush. If the superficial erythema 
faded within a few hours, the reaction was considered to be nega- 
tive. If, however, within twenty-four hours there occurred a well- 
circumscribed area of erythema, with infiltration and papular 
elevation of the skin surrounding the point of puncture persisting 
for twenty-four hours or more, the reaction was called positive. 
During the course of pneumonia, Weil found the reaction to be 
negative, but twenty-four hours to two weeks after crisis a con- 
siderable proportion of patients reacted positively. Normal indi- 
viduals and subjects suffering from diseases due to organisms 
other than Pneumococcus might or might not exhibit a reaction. 
Analogous results were reported by Steinfeld and Kolmer 
(1917), 1315 who employed saline suspensions of washed organisms 
of Types I, II, and III heated to 60°. A positive reaction consisted 
in a definite papule with an area of erythema greater than one 
centimeter in diameter accompanied by slight edema. Positive re- 
actions were observed in 30 per cent of patients suffering from 
lobar pneumonia, but consistently negative results were obtained 
with normal individuals and with those afflicted with various 
chronic diseases. All positive reactions noted occurred after crisis 
or after infection had persisted for a long period of time. Stein- 
feld and Kolmer stated that there was no constant relation be- 
tween reactions to the protein of the various types of pneumococci 
used and the types found in the sputum. Weiss and Kolmer 
(1918) 1510 employed as antigen hemolytic toxin prepared by dis- 
solving washed, living pneumococci in sodium choleate. Among 
adult patients with lobar pneumonia the intracutaneous reaction 
was elicited as early as the fifth and as late as the thirteenth day 



462 BIOLOGY OF PNEUMOCOCCUS 

of the disease (two days before and six days after crisis). In chil- 
dren the reaction was demonstrable at about the same time, but 
was negative immediately following crisis or within one or two 
days after crisis. When the pneumonia resolved by lysis, the pa- 
tients reacted as late as the thirty-second day. In general, the 
test was positive in all active cases but was negative in all control 
patients whether children or adults. Weiss and Kolmer considered 
that the phenomenon was a manifestation of a state of allergy to 
toxin but, since the reactivity of the skin was present during the 
stage of toxemia and disappeared during convalescence, it is diffi- 
cult to look upon the reaction as being strictly allergic in nature, 
at least in so far as toxin is concerned. 

Bigelow (1922), 115 employing saline and aqueous autolysates of 
pneumococci as antigens, succeeded in evoking type-specific dermal 
reactions in some cases, while in others and in normal controls a 
reaction occurred differing in character from that resulting when 
the type of autolysate and of the infecting organism were homolo- 
gous. Larson (1926) 787 also reported irregular results of skin 
tests obtained in normal persons and pneumonia patients injected 
with pneumococcal filtrates. He apparently assumed that the reac- 
tion was due to toxin since allergy was not mentioned in the com- 
munication. 

The question became somewhat more confused by the observa- 
tions of Herrold and Traut (1927). 637 The majority of pneu- 
monia patients reacted negatively to filtrates of broth cultures of 
Pneumococcus, whereas nearly all normal individuals similarly 
tested also gave negative reactions. By repeated tests on pneu- 
monia patients it was found that the condition preventing a reac- 
tion developed early in the disease and usually persisted through- 
out convalescence. The reactive ability of the individual corre- 
sponded to the capacity of the serum to neutralize the active prin- 
ciple of the filtrate, the serum of a non-reactive subject usually 
effecting a partial or complete inhibition of the antigen. 

Comparable to the results of Herrold and Traut were those of 



HOST RESPONSE TO ANTIGENIC ACTION 463 

Kramar and Gyiire (1929), 754 Injecting intradermally suspensions 
of pneumococci of the different serological types, the authors 
noted positive skin reactions in only 18 per cent of pneumonia pa- 
tients but in 50 per cent of healthy individuals. In the case of the 
pneumonia patients reactivity of the skin appeared in eight to 
sixty days after resolution of the pneumonic process. The reaction 
was, in general, type-specific for Types I, II, and III, as judged 
by the serological relation of the antigen employed to the organ- 
ism isolated from the sputum of the subject tested. 

Contradictory results were described by Tillett and Francis 
(1929), 1408 who found that pneumococcal polysaccharides, when 
injected intradermally into patients convalescent from lobar pneu- 
monia, were capable of eliciting a definite response. A patient's 
ability to react was apparently associated with both recovery from 
infection and the presence of type-specific antibodies in the cir- 
culating blood. In addition to the action of the polysaccharide, 
the so-called nucleoprotein of Pneumococcu?, when similarly in- 
jected, caused a local cutaneous reaction in subjects during con- 
valescence from pneumonia. The local lesion resulting from the in- 
jection of protein was tuberculin-like in character, and differed 
from that evoked by type-specific polysaccharides in gross ap- 
pearance, time of development, and duration. Although the pres- 
ence of precipitins reactive with pneumococcal protein could be 
demonstrated in individuals acutely ill with or convalescing from 
pneumococcal pneumonia, the concentration of antipneumococcic 
antibodies in the blood serum appeared to be unrelated to the pa- 
tients' capacity to react to intradermal injection of the protein. 

In a second communication, Francis and Tillett 478 amplified 
their observations on the appearance of cutaneous reactions dur- 
ing lobar pneumonia. Immediate wheal formation and erythema 
followed the intradermal injection at the time of crisis of homolo- 
gous polysaccharide in 100 per cent of Type I patients, in 55.8 
per cent of those infected with Type II Pneumococcus, and in 44 
per cent of those infected with pneumococci of Type III. A de- 



464 BIOLOGY OF PNEUMOCOCCUS 

layed reaction ensued after a similar injection of pneumococcal 
protein. The appearance of circulating antibodies for one or more 
heterologous types in a group of patients repeatedly tested with 
soluble specific substance was believed by the authors to be refer- 
able to the antigenic action of the polysaccharide introduced into 
the skin during successive tests. 

In opposition to the conception that skin reactivity to the cap- 
sular polysaccharide of Pneumococcus is associated solely with 
recovery from pneumonia are the results of Alston and Lowdon 
(1933). 10 The majority of the individuals studied (63 per cent) 
who had not recently suffered from pneumococcal infection reacted 
positively to the intradermal injection of Type II polysaccharide, 
the percentage of reactions increasing in the older age groups. Re- 
tested five to twelve months later, 92 per cent of the subjects pre- 
viously exhibiting a positive reaction were found to have retained 
the ability to react to the same antigen. The capacity to respond 
to similar injections decreased and in some cases entirely disap- 
peared when the test injections were repeated at short intervals. 
The authors also reported that a considerable number of normal 
subjects, especially older persons, exhibited a delayed reaction in 
addition to the immediate reaction or independent of it. 

The manifestation of both immediate and delayed reactions may 
be explained by the fact that the introduction of pure pneumococ- 
cal capsular polysaccharide into the skin of a positively reacting 
subject is promptly followed by a local inflammatory condition, 
while after the similar injection of the C Fraction or of somatic 
protein, an interval elapses before any local reaction occurs. Skin 
sensitivity to pneumococcal type-specific carbohydrates and the 
presence of specific antibodies in the circulating blood were found 
to exist independently of each other. The findings of Alston and 
Lowdon agree with those reported in the previous year by Sutliff 
and Finland, 1360 who reported that skin reactions to acetic acid- 
precipitable protein and autolysates of Pneumococcus were nega- 



HOST RESPONSE TO ANTIGENIC ACTION 465 

tive or rarely positive in infants, infrequently positive in child- 
hood, and positive in a high percentage of adults. 

Finland and Dowling (1935) 442 performed skin tests on pa- 
tients recovering from lobar pneumonia and also on normal indi- 
viduals and persons suffering from non-pneumococcal affections. 
The antigens employed were the capsular polysaccharides of pneu- 
mococci of Types I, II, and III, and the cellular carbohydrate of 
Wadsworth and Brown. Immediate cutaneous reactions, similar 
to those described by Tillett and Francis, were elicited in patients 
recovering from pneumonia. These reactions were, in general, 
type-specific and were associated with the homologous antibody in 
the serum. Similar reactions were obtained with preparations of 
soluble specific substance and cellular carbohydrate prepared from 
the same pneumococcal types by the methods of Heidelberger, 
Goebel, and Avery and those of Wadsworth and Brown, respec- 
tively. 

Characteristic delayed reactions were seen only with the cellu- 
lar carbohydrate and were not associated with type-specific anti- 
bodies. The delayed reactions were most frequently observed fol- 
lowing the injection of the cellular carbohydrate of the atypical 
Type I Pneumococcus and of the analogous antigen prepared 
from the virulent Type I strain. Delayed cutaneous reactions to 
the cellular carbohydrate of an atypical Type I Pneumococcus 
were obtained regularly during the febrile stage of a variety of 
infectious diseases but could not be evoked soon after recovery in 
these cases. The results are similar to those obtained by Francis 
and Abernethy 475 with the somatic C Fraction of Tillett and 
Francis. 

Among the non-pneumonic subjects on whom observations were 
made, immediate reactions in general were uncommon. They oc- 
curred most frequently with the cellular carbohydrates of Type I 
and atypical Type I pneumococci. Delayed reactions with soluble 
specific substance of any type did not occur, but they were fre- 



4,66 BIOLOGY OF PNEUMOCOCCUS 

quent with the cellular carbohydrates of Type I and atypical 
Type I organisms. 

In the group of subjects who gave a previous history of pneu- 
monia, none of the eight tests done with capsular polysaccharide 
was positive. A higher incidence of positive, delayed reactions was, 
however, obtained in the small number of subjects of this group 
tested with the cellular carbohydrates. 

ACTIVE AND PASSIVE SKIN ALLERGY 

The lack of uniformity in specific cutaneous reactions occurring 
in normal human beings and in those ill with or convalescing from 
lobar pneumonia was encountered by Cecil and Austin (1918) 204 
in following the administration of vaccines made from pneumo- 
cocci of Types I, II, and III and used for prophylactic injection 
of healthy men. Small, sterile infiltrations, disappearing spontane- 
ously, occasionally followed the injection of vaccine, and similar 
lesions appeared in the same individuals after subsequent injec- 
tions. Subjects exhibiting these reactions rarely possessed any 
notable amount of agglutinative or protective antibodies in the 
serum after vaccination, nor was the reactive property specific 
for any type of Pneumococcus. 

Gutfeld and Nassau (1926) 580 treated nurslings ill with broncho- 
pneumonia with vaccines composed of heat-killed pneumococci, 
and injected normal children or those ill from some cause other 
than bronchopneumonia intracutaneously with vaccine or with 
watery autolysates of the same cultures from which the vaccines 
were prepared. The development of a reddened papule was ob- 
served at the site of puncture, appearing within eighteen or 
twenty-four hours and disappearing after forty-eight hours. No 
child under one and one-half years of age gave a positive reaction, 
nor did any of the young patients suffering from bronchopneu- 
monia. The reactions occurred in more than one-half of the nor- 
mal individuals tested and in a like proportion of those not af- 
flicted with the disease. When the autolysate was mixed with serum 



HOST RESPONSE TO ANTIGENIC ACTION 467 

of non-sensitive children no reaction took place, whereas the serum 
of sensitive children failed to neutralize or inhibit the action of the 
antigen. Gutfeld and Nassau assumed that individuals failing to 
react to the intradermal test possessed in the skin or in the serum 
a specific antibody similar to antitoxin, inherited in the case of 
young nurslings or developed during an attack of bronchopneu- 
monia and then later lost. 

Jamieson and Powell (1931) 676 believed that pneumococci pro- 
duce toxic substances not unlike those obtained from various 
streptococci. The substances could be detected and measured by 
skin tests on human subjects and by similar tests performed on 
rabbits of a suitable breed. Serum yielded by horses that had been 
treated by subcutaneous injections of the toxic substance pos- 
sessed the ability to neutralize the skin-reactive property of the 
toxic antigen. In a second paper, Jamieson and Powell reported 
the development of positive reactions when filtrates of pneumo- 
cocci of Types I, II, III, and IV were injected intradermally into 
large rabbits. Similar reactions to filtrates of all four pneumo- 
coccal types were also observed in the case of twelve normal hu- 
man subjects, who exhibited a degree of sensitivity approximately 
one hundred times that shown by rabbits. The toxic filtrates could 
be neutralized by the special immune serum developed in horses by 
the injection of the same antigen. 

The work of Coca 245 bears certain resemblances to that of 
Jamieson and Powell. 676 Filtrates of pneumococci exhibiting toxic 
action for young children when injected subcutaneously also in- 
duced local inflammatory reactions in the skin at the site of punc- 
ture. In Coca's first experiments the filtrate was introduced into 
the child's skin and at the same time a considerably larger dose of 
the filtrate was injected subcutaneously. Subsequent skin tests per- 
formed with the filtrate on the children so treated were attended 
by a diminished reaction. Treatment of the subjects with a toxoid 
prepared by heating the filtrates with formalin tended to reduce 
the number of positive reactions. When applied to adults giving no 



468 BIOLOGY OF PNEUMOCOCCUS 

history of previous pneumonia, the test was positive in 56 per cent 
of the subjects tested. In the case of individuals having a history 
of pneumonia in the past, 15 per cent of the subjects reacted, 
while in convalescent patients only 5 per cent exhibited positive 
reactions. Coca reported that not all filtrates tested were anti- 
genic and, at the time, ventured no statement concerning the type- 
specificity of the phenomena. 

Recently Coca, 246 after a further study of the alleged pneumo- 
coccal toxin, stated that the action of the toxin in the skin was 
type-specific and, furthermore, that the toxin was not the type- 
specific polysaccharide. 

The application of the skin test as a guide to serum therapy 
and as a prognostic aid was suggested by Francis (1933). 473 All 
but one of the patients who had recovered from Type I Pneumo- 
coccus pneumonia (the great majority of them had received spe- 
cific antipneumococcic serum) gave an immediate reaction to the 
Type I polysaccharide at about the time of recovery. Francis 
claimed : 

The test has distinct advantages over the agglutination reaction in 
that it is not merely an index of circulating antibodies. When positive, 
it invariably denotes that recovery has begun; when negative, it indi- 
cates further serum therapy. The mechanism of the positive skin test is 
closely related to that operative in recovery from pneumonia, and is ap- 
parently the resultant of antibody and tissue activity. 

THE MECHANISM OF DERMAL ALLERGY 

In addition to the foregoing discussion, two studies may be 
mentioned which have a direct bearing on the physiological or 
immunological processes operating in the production of the aller- 
gic state and in the manifestations of the hypersensitive condition 
as elicited by pneumococcal derivatives. Martin and Hill (1930) 866 
tested the effect of an alien protein on the reactivity of dermal tis- 
sues in albino guinea pigs receiving intradermal injections of liv- 
ing pneumococci. One group of animals received into the skin of 



HOST RESPONSE TO ANTIGENIC ACTION 469 

the left flank an injection of a suspension of washed pneumococci 
followed immediately by a similar injection of milk protein on the 
right flank. Forty-eight hours later they were given on the left side 
at some distance from the first puncture an injection of the same 
organism heated for thirty minutes at 60° and again milk protein 
into the skin on the right flank. The second group of guinea pigs 
were treated in the same manner but without the injection of milk 
protein. Examination of the animals of the second group revealed 
an area of induration and inflammation at the site of the injection 
of pneumococci, whereas the animals that had received milk pro- 
tein in addition to the pneumococcal suspension showed only the 
slightest response to the bacterial antigen and none to milk pro- 
tein. When intramuscular injections of milk protein were substi- 
tuted for intradermal administration, with all other conditions of 
the experiment the same as before, the reactions of the animals so 
treated were less pronounced than those in the guinea pigs receiv- 
ing only pneumococci, but more marked than those occurring when 
the protein was injected into the skin. While it is apparent that 
the injection of foreign protein inhibited the reactivity of the cu- 
taneous tissues to the injected suspension of heated pneumococci, 
the significance of the experiments is not clear. 

Kramar and Gyiire (1930) T56 sought other means for learning 
the manner in which the allergic manifestations of the skin take 
place. A comparison was made of the bactericidal, bacteriotropic, 
protective, and antitoxic action of the serum, plasma, and defibri- 
nated blood of human beings who reacted either positively or nega- 
tively to an autolysate of Type II Pneumococcus. From the 
experiments there appeared to be no correlation between the con- 
tent of humoral antibodies and the outcome of the skin tests. The 
authors then studied the relation of the skin reactions in man 
evoked by pneumococcal autolysate with the so-called "pharmaco- 
dynamic reaction" of Hecht and von Groer, which consisted in de- 
termining the maximal dose of epinephrin and caffein-sodium sali- 
cylate which, when injected alone into the skin, would produce 



470 



BIOLOGY OF PNEUMOCOCCUS 



twenty-four hours later no redness at the point of injection. For 
the details of the experiment the original communication of Kra- 
mar and Gyiire should be consulted. The results led Kramar and 
Gyiire to conclude that the cutaneous reaction to pneumococcal 
autolysate was similar to that produced by tuberculin and the op- 
posite of that caused by diphtheria toxin and, therefore, that the 
pneumococcal skin test was an allergic reaction and was an ex- 
pression of cellular immunity. 

In a theoretical discussion of the mechanism of skin reactivity 
to Pneumococcus, Kramar and Gyiire presented the theory that 
allergy represents a labile condition of the animal organism, since 
artificial immunization causes positive reactivity to become nega- 
tive reactivity, while colds, exhaustion, fatigue, massive infections, 
and diseases other than pneumonia, and possibly the age of the 
individual, change the state of allergy to one of anergy. Kramar 
and Gyiire presented the following schematic representation of the 
factors concerned in the three hypothetical states prevailing in 
man in relation to hypersensitivity to Pneumococcus. 



Anergic phase — no 

protection 
Skin test — negative 
New born subjects 
The number of nega- 
tive reactors de- 
creases with age 



II 

Allergic phase — labile 
immunity 

Skin test — positive 

The majority of chil- 
dren and adults 

The number of subjects 
increases with age 
among nurslings 



III 

Fastness — stable im- 
munity 

Skin test — negative 
The minority of chil- 
dren and adults 
Those artificially 
immunized 



The authors explain that shifts in phase to the right are influ- 
enced by increasing age of infants, by recovery from pneumococcal 
infection, and by repeated slight infections such as common ca- 
tarrh of the upper respiratory passages. Reverse shifts to the left 
are brought about by the debilitating causes already mentioned. 
The foregoing presentation of the subject of dermal allergy to 



HOST RESPONSE TO ANTIGENIC ACTION 471 

pneumococcal products is bewildering, and from the data as pre- 
sented it is difficult to gain a clear conception of the true signifi- 
cance of specific skin tes