CHAPTER V

PNEURIOCOCCAL DISSOCIATION AND
     TRANSFORMATION

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

T HE constancy of the biologicrL1 characters of I'neumococcu's
  depends on the conditions of its surroundings. When the con-
ditions are favorable, the morphological and serological integrity
of the ccl1 remains st,able. Sul>jcctc~l to unfnvornhlc influences,
I'nrumococcus exhibits great lability of form and function. The
form of the cell mny pnss through every stage from the typical en-
capsulated diplococcus to one completely denuded of capsule;
pnthogenicity mny be diminished from full virulence to entire ab-
sence of infectivity; and antigenic tend serological properties mnp
lose strict type-specificity and rctnin only the bronder spccies-
spccificit,y. With the restoration of favorable conditions, the deg-
radation process, if it has not proceeded too far, tenses and is re-
versed, the cell ngnin assuming its typical characters. But, what is
still more rcmarknblc, 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 dissociat.ion, the
actual transformation of pneumococcal types has been espcri-
mentally induced and, possibly, species mutation has occurred.
I,ack of knowledge of the existence of bncterial variants and of the
factors inducing dissociation has undoubtedly caused much confu-
sion in bncteriological diagnosis and in the interpretntion of the
serum reactions of Pneumococcus.

  Enrly Obserzntion~s of Dissociation,: l&91-1981
Bacterial variation is not a new phenomenon. It is only the
study and the csplnnution of the underlying causes thnt are recent.
In 1801, Krusc and I'ansini'"?' first called attention to changes in
morphology, cultural clinrncters, and virulence of pneumococri u11-
dcr artificial cultivation. Pure cultures freshly isolated from pncu-
manic material were typical in appearance durmg early gcnera-
tions but, on continued cultivation on unfavorable media, the cells
exhibited deviations from the normal charncters. The lnnce-shaped,
diplococcal forms became strcptococcal or even bacillary, the cnp-
sule rapidly vanished, and virulence waned. The degenerated cocci,
when passed through susceptible nnimnls, regained their capsules
nnd virulence, and when returned to favorable media, ngnin showed
1ionm1 1)nc'umococcaI ~riorpl~ology. Krusc and Pansini, therefore,
noted many of the fcntures of dissociation and were awnrc of the
first cnuscs of the phenomena.
In the nest year, Arkharow" reported chnngcs taking place in
cultures cultivntcd in the serum of vaccinated animals. Growth
wns slow in developing nnd at the end of three or four days the
cocci began to diminish in size, to grow m long chams, and to lose
virulence. Four years later, Eyre and \Vashbourn373 described the
varintions 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 froni the parent stock.
It, in fact, represented a distinct variety, possessing practically
no virulence, and growing luxuriantly, even at 20oC., on all the
usual media." The first attempts to induce this variant to revert to
its former stnte were unsuccessful. Then, by pnssagc through a
rabbit, the vnriant reverted to its original form.
Hiss, Borden, and Knapp ( 1905)051 encountered organisms, in-
distinguishnble in fcrmentativc reactions from pneumococci, which
showed variations in morphology or agglutination, and the nu-
thors considered thnt 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
discovcrics, was that of Rosenow" 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 hcmolysis 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 &o-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 FrieYQ4 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 Friedllinder's bacillus, Pas-
tcurella, and Pneurnococcus. The same effect was produced by cx-
posing the organisms to immune serum; while the reverse process
took place when "pianticatcd" bacteria were grown in normal se-
rum--they regained their resistance to phagocytosis.*
In the next year, Stryker'348 described the variations induced in

o Neufeld and Schnitzer credited the first demonstration of bacteria% dis-
sociation to Friel, although Hadicy ascribed the discovery to Baerthlein
(1912). The phenomenon had, however, been observed much earlier by Kruse
and Pansini (1891),X3 and by Arkharow (1892),17 and hnd been described in
some detail by Eyre and Washbourn in 1897.

P

    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 antigcnic
properties. Reversion of the changed forms to the original t,ype
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 pncumococci 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), I8 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,"" 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 arc avirulent. Cocci of the R
colonies may revert to the S form, or they may rcmuin stable for
gcncrations. 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 Pncumococcus from the viru-
lent, complex type of S cell to an attenuated form with a simpler
antigcnic 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-
mcndcd 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. Kc suggested as an explanation of the change the view
that serum might disorganize the biological functions of Pncumo-
coccus by precipitating the capsule, thus inhibiting the sccrction
of antilcucocytic substances and rcndcring the orgnnism tempo-
rarily harmless, and that when pncumococci divided in the animal
body in the prcscnce 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-
woodZ4* contributed an interesting theoretical discussion. It would

     DISSOCIATION AND TRANSFORhlATION      139

take us too far afield to quote from it here, but the communication
is recommended to those rcadcrs 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,"``-' by the substitution of normal dog
blood for rabbit or horse blood in agar for plate cultures, obtained
such marked difrcrenccs in the morphology of S and It colonies
that differentiation, they claimed, became estrcmcly simple. With
a moderate degree of magnification (X 28), the S colonies were
seen to bc 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 hc-
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 belicvcd 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 (1023),l" in conducting experiments similar
to those of Strykcr, also found that growth of pneumococci in ho-
mologous serum result,cd in a marked loss of virulence, accom-
panied by constant and distinct changes in agglutinative proper-
tics with respect both to the character of agglutination and the

x   zone of optimal reaction. The authors found the changes not to bc

L   a gradual alteration of nil members of a culture but, instcud, there
  appeared to bc a comparatively rapid and complete change in in-

diridual 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,

i
i   and C, could be distinguished.
b    In the same year, analogous observations were published by
c    Yosliioka.`"B4 Typical pneumococci underwent, apparently regular

6   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, agglutinablc by anti-
strcptococcic 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 Giittingen in 1924, Neufelds78 reported a change in bilc-
solubility of pncumococcal variants, as well as in their suscep-
tibility to optochin. In the same year, Fclton and Dougherty4*'
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

Amossl' 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 I90 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-

F'

     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-

i  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-

1  nins but no protective antibodies for the parent strain. It seems

1  clear from AIIIOSS' experiments that he had succeeded in effecting a
permanent degradation of a virulent Type I Pncumococcus, with a
  loss of type-specificity, but not of species-specificity.

The results of Reimann's"*" study, published in the same month
in which Amoss' publication appeared, agreed with both Amoss'
and Griffith's observations. Reimann reported that cultures of
pncumococci 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 fnom 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, Rcin~ann1126 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 varinnt
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 scra produced by immunization with S strains may contain spc-
ties-specific antibodies in addition to those which are type-specific.
Each kind of antibody can be removed separately from these sern by
selcctivc 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 Hcidelbcrger of the anti-
genie chemical constituents of the pneumococcal cell.
. `J!akan~i,`373 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.

Takann'3'5 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
artificinl media. The explnnntion offered wns thnt in the body the
organisms lose their old receptors and acquire new ones. Takami
separated five typical strnins of "culture-bacteria" (pneumococci
long grown on blood agar) into colonies that were still markedly
agglutmable, 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 Meye?l 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 atypicnl R forms with divcrgcnt cultural and serological char-
acters. The espcrimcntnl data, however, are insufficient for judg-
ing the claim.

For determining the true character of normal strains and of dis-
sociants, Schien~nnn'*26 adopted as a criterion the possession of a
type-specific (dominant) hapten as a prerequisit,e 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 hnptcn; sec-
ond, the repression of cross-ngglutinntion in heterologous serum ;
and, third, mouse virulence. According to these standards, in


144       BIOLOGY OF PNEUhlOCOCCUS

addition to normal and degraded R forms, Schicmann 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.

ELECTILOPHORETIC POTENTIAL OF VARIANTS

Falk, Jacobson, and Gussin,s83 and then Falk and Jacobson,3So
studied another criterion for variability. The authors measured
the electrophoretic potential of Blake's variants A, 13, and C from
Type I I'neumococcus 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*oo-' and shortly
afterward by Morgenroth, Schnitzer, and Berger,"' 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
Schnitzcr-Berger medium.

DISSOCIATION AND TRANSFORMATION      145

In Amzel's" 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) ,e74-6 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 elcctrophoretic 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. LevinthalsoO also observed changes in virulence and in
the cultural nnd 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 viva wcrc de-
scribed by Wadsworth and Sick1es.l"' 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 caps& formation, antigcnic
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,`*" in a later study (1932) of I meumococcal 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 42O, and survived even
after incubation at 43.5'. No other pneumococcal strains studied
were alive after twenty to twenty-four hours at 42'.

That l'neuniococcus may, however, retain its specific type char-
acters when growing in the animal body was proved by Mcgrail
and EckcP' 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 agglut,inability. Here the conditions differed from
those in the horses harboring pneumococci, as reported by Sic-
kles,`2'0 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.

Reimann1128 found that R forms occurred in ZCVO but could dis-
cover in his experiments no positive evidence that recovery from
p~wu~nococcnl 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 study11Z9 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 serutn.
Rcimann considered that recent experimental studies indicated
that virulent S pneumococci might dissociate into the R form in
viva; that R forms occasionally could be found in the sputum of
pneumonia patients, and also might live dormant in viva for a con-
siderable period of time.

The recovery of R variants from the body has recently been re-
ported by Shibley and Rogers.1*62 Twenty-four lung punctures
made in lobar pneumonia patients at the time of crisis or lysis
yielded It forms of pneumococci in all but four cases.

DETAILS OF COLONY POIlMATION

DawsonZg9 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 trnnsfers, 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 prescncc 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 It and S forms inter-
ested Paul (1927) ,108' 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, Pau1'068 gave further information conccrn-
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 I'neumococcus, Atkin*' 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.

GrumbachGe3 also studied the details of colony formation accom-
panying the varying degrees of pneumococcal dissociation. He dif-
fered with Atkin,*' 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
hcmolysis 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 t,he classical picture
of pneurnococcal colonies. The thickness of the peripheral ring va-
ried, and in one case there was a "wall" formation of the type
Bucrger and Ryttenbergl" claimed to have found solely in colonies
of streptococci. Grumbach also described a "wing-form" colony
which he believed to bc similar to that supposed to be caused by
phage action on streptococci, and concluded that the same colony


150        BIOLOGY OF PNEUhlOCOCCUS

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

FaragG (1832) 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 D awson.

ANTIGENICITY OF ROUGII FORMS

  Tillctt'"06 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'404-B
had previously shown that a similar immunization treatment in-
duced active resistance to Type III infection.) Furthermore, the
whole titrated blood of the immune rabbits passively protected
' normal rabbits against infection with Type I and Type III pncu-
mococci, but failed to confer a like protection on mice. According
to Till&t this form of acquired resistance to pneumococcal infec-
tion elicited by R organisms devoid of type-specificity, and cxcm-
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

o For n full discussion and bibliography of microbic dissociation up to that
time the reader is referred to Hadley'ss84 comprehensive article.

      DISSOCIATION AND TRANSFORMATION     151

changed respiratory capacities of pneumococcal variants. Accord-
ing to Finkle's440 measurements, the capacities of organisms of
Types I and II were altered during conversion from the S to the R
form. For Type I I'neumococcus 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.
Pncumococcus 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 0, 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,,). When compared with
the oxygen consumption of animal tissues, Type II strains con-
sumcd over twenty times as much oxygen as did isolated rat kid-
ney tissue, and almost one hundred times as much O2 as isolated
dog muscle.

A respiratory phcnomcnon connected with loss of virulence has
been described by Sevag and Maiweg."" 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 Scvag 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


16.2        BIOLOGY OF PNEUMOCOCCUS

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

Petrie ( 1932)`os" suggested one more means for the idcntifica-
tion of R and S variants. In stab cultures in agar plates contain-
ing 5 per cent immune serum the virulent S pncumococci 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
pncumococcal 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. Strum 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)730
mentioned intermediate forms growing in colonies dcsignatcd as
SU and RK. In other characters, however, the strains were either
true S or R forms. Klumpcn recognized the Fliitterformen de-
scribed by Grumbach, and noted that the organisms comprising
daughter colonies wcrc of the smooth type.

Still other variants intermediate between the S and R forms
were derived from pneumococci by Blake and Trask (1933) .13" By
growing Type IS pncumococci 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 Ib and
Type Ic, wcrc easily stabilized in pure culture. All showed an or-
derly change in agglutinative reactions in homologous and hcter-
ologous immune serum, and also underwent a progressive loss of
virulence for mice. Blake and Trask produced only one intcrmcdi-
ate form from Type IIS and none from Type 111s.

The importance of recognizing intcrmrdiate variants in t,hc dis-
sociative process was emphasized by Paul'oGD of Blake's labora-

DISSOCIATION AND TRANSFORMATION     153

tory. He believed that two methods of inducing dcgradative 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 grcatcr 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, `*' 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+I935).345-" 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-
genie 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 metbemo-


154        BIOLOGY OF PNEUMOCOCCUS

globin in blood. Without an actual visual comparison of these V
rarinnt,s with the principals and intermediates dcscribcd 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 polyphnsic cycle in bac-
terial dissociation.*

REVERSAL OF I~ISSOCIATION

Griffitli"G2 was successful in reversion experiments in the animal
body. Some R strains which had not entirely lost their soluble spc-
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 agcing 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 Avery304 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 scrum.t
The authors failed in a similar attempt with a Type IR culture. As
in mouse passage, reversion by cultural methods was accompanied

o Rakietenrlla believed that a peculiar organism obtnined from the peritoneal
fluid nnd heart's blood of a mouse after inoculation with a highly virulent Type
II Rneumococcus was n pneumococcal variant. It was a Gram-positive hncillus,
bile-soluble, agglutinated with Type II serum in a dilution of 1 to 4.00, 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 Ruids rnises douht as to its true pneumococcnl origin.
t Compare Soule'slsos similar results with Blncillus subtilis (1927).

     DISSOCIATION AND TRANSFORMATION      156

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 UY 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 bcforc 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
GO', 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.

o ?oo??*? ?o????? 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, mutinous colonies of Type III. The newly devcl-
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 pncumococci 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 it 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 pncumococcus 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 WC 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 cx-
planation for the many baffling problems encountered in the study
of the spread and the invasiveness of pncumococci, and of the clini-
cal pathology of pneumococcal infections, not to mention the
broader bearing on the many riddles of microbiology.

Ncufeld and Levinthal""' 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. Ncu-
fcld and Lcvinthal thus converted an avirulcnt 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."" 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 pncumococci; IIR became IS or
IIIS, 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.

Baurllcnn's83 efforts at reversion (1932) were more fruitful than
Rcimann's. By subculturing R strains with homologous and heter-
ologous vaccines consistin, v of heat-killed cultures, he changed the
R strains into their original S forms and to the S form of a diffcr-
cnt type. Baurhtnn inclined to Griffith's view that all pncumococ-
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. Baurhcnn claimed to be t,he 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)298 confirmed and expanded Griffith's observa-


I58        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 hcterologous S
types to revert to the type of the vaccine ; S vaccines heated fifteen
minutes at temperatures between 80" and 100o were not active in
causing R variants derived from hetcrologous 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 SO0 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 hetcrologous types to revert to the type of the
vaccine employed. Dawson successfully converted a single-cell R
strain derived from a Type 111s pneumococcus into a Type IIIS,
a Type IS, and a Group IVS organism. On the other hand, every
attempt to produce transformation of type in Z&JO failed.

TRANSFORMATION BY VACCINE AND ANIMAL INOCULATION

In 1030, Dawson and Sia3"' announced the transformation of a
Type IIR into a Type 111s pncumococcus. 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 ant,i-R serum and of blood broth. 1f'hcn the activating organ-
isms were heated for fifteen minutes at loo", 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.

     DlSSOCIATION AND TRANSFORRlATION      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 GO" 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 dcvclopment of intermediate stages was de-
scribed by Dawson and WarbassesoO in 1031. The original culture
was a virulent, single-cell isolation of Type II Pncumococcus. One
drop of a 10-O dilution of the culture was scedcd into a medium
containing homologous immune serum together with large quanti-
ties of Type III p neumococcal 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
pncumococcus can bc transformed into other type-specific S pncu-
mococci by growth in homologous immune serum in the presence of
hetcrologous vaccine. Although the conditions of the experiment
were unfavorable to the dcvclopmcnt of R forms, the authors
thought it was probable that the organism ncvcrthelcss passed
through this intermediate stage during the transformation.

In 1931, Sin and Dawson'272 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)" 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), All away' prepared active and spe-
cific extracts by dissolving S pneumococci in sodium desosycholatc
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 reprccipitation 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 SO" 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 wit,hout the addition to
the culture-extract mixture of blood serum or of ascitic or pleural
fluid.


     DISSOCIATION AND TRANSFORMATION     161

         Dawson Classification
A dissociation form, other than the S and R forms, was de-
scribkd in 1934 by Dawson."' 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"`en 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 incuba!ed 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 althougb it is emphasized that the process is both gradual


162       BIOLOGY OF PNEUMOCOCCUS

arid continuous. In the first stage, wbicb may suitably be termed RI, tile
colonies present more or less tlie general appearance of smooth (old tcr-
minology, "rough") colonies but the surface is more coarsely stippled.
The constituent organisms are more or less typical pneumococci sbow-
iug a tendency to staphylococcal grouping and occasioual swollen or
club forms may be seen. In the second stage, R2, tile colonies present a
still rougher appearance and the outline may appear slightly irregular.
`Ibis irregularity frequently becomes quite pronounced after several
days' growth The bacteria in this stage are much more pleomorphic
aud are frequently elongated in an extreme lanccolate manner. They
still retaiu Gram's stain. In the third stage, R3, the surface of the col-
ouy becomes exceedingly rough and tile 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 dipbtheroid
clernents 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
piieumococcus. The fourth stage, R*, can only be defined wit11 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
CR">.

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

Dawson dcscribcd the It * S change in which intermediate forms
of the type seen iI1 the S * 1% 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 sclcctcd. 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 point,ed out certain discrepancies in the character-
istic features of the S and It forms as dcscrihed by Griffith for
PI~C~~IOCOCCIH and those described by Arkwright for the colon-
tvl'lioid-dyscrltery 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 Pncumococcus while
other new distinctions did not have a place in Arkwright's original
descriptions. Dawson has portrayed these diffcrcnccs in tcrmi-
nology in a diagram which, although as yet unpublished, was
kindly loaned to the authors.

I     NOT
  DESCRIBED   M    S

2 s

31 R

M

S

  NOT
DESCRIBED  R

Courtesy of Dr. M. A. Dawron

ILELATIONSHIPS OF MUCOID, SMOOTH, AND ROUGH COLOXIES
   OF BACILLI OF TIIE COLON-TYPHOID-DYSEhTERY

          GROUP AND PNEUMOCOCCUS


164       BIOLOGY OF PNEUMOCOCCUS

When Dawson compared the salient characters of the three
pncumococcal 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, form of Friedlander
are similar to the smooth form of bacilli of the enteric group ; while
Dawson's new variant and Julianelle's R, 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 pncumococci as follows: Mucoid or M would re-
place the present smooth or S ; smooth would be substituted for the
former rough (R, form of Friedlander bacilli) ; while rough or R
would be applied to the new variant described by Dawson and the
R, form of Friedhinder'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 bctwecn 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     166

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. Hadlcy's opinion expressed in a letter written in 1933 to
Dawson was in part :*

g    Making a decision regarding the proper course to pursue in changing
  the nomenclature now employed for designating the phases of the pneu-
i
:,   mococcus, in favor of the symbolization which your studies thus far
F   seem unquestionably to justify, might easily depend on how fully an in-
i   vestigator has in mind the details of dissociative variation as a phe-
5   nomenon observable in all bacterial species, and how clearly he can
  perceive the parallel trends in such variations,-as opposed to a limited
L   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
7
8   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

c   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-
t

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 afithors appreciate the courtesy of Doctors Hadley and Dawson in
granting permissiou to include portions of this letter here.


1GG       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 aff;iirs 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," whencrer 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-
solutcly 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 bcIicvcd 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 Strcptococ-
cus haemdyti~crcs. From the latest study by Dawson,""3 it would
seem that he succcedcd 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
Pncumococcus. 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
t,hc phenomenon of bacterial variation in a wide variety of bac-

DISSOCIATION AND TRANSFORhIATION      167

terial spccics fit. 9 into a IIIOI'C or less orderly pattern." This pat-
tern, bcsidcs fitting bacilli of the colon-typhoid-dyscntcry group,
the types of B. fricdhdcri, and probably the streptococci, would
bring order in the arrangcmcnt of the many variants of pneumo-
cocci that have been described under a wide diversity of terms.
Thus, the modifications A, 13, and C of Schnitzcr and Bcrger,
Blake and Trask's intermediates Type I a, b, c, d, and C, Fj'ads-
wort,li and Sickles' atypical strains, Reimann's daughter-colony
variants, the "wall" type of Bucrger, the Fliittcrformen of Grum-
bath, possibly the P-C or phantom colonies and the smooth N and

I,   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 RI< dissociants of Klumpcn, and of
  course the R and S forms of Griffith, and the new variant of Daw-
son might conceivably bc arranged in accordance with the general
pattern and would all either fall into the chief places designated by
  Dawson's RI, S, and R or into the spaces between these prcdomi-
  nat,ing forms.

   The scheme of Dawson, therefore, revolutionary as it may seem,
;.
P   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
  arc full of new meaning to the bacteriologist, biochemist, immu-
  nologist, and particularly to the physiologist. They prove that
  PI~C~~OCOCCU~ has the potential ability to synthcsizc simple sugars
  into diverse, complrx, and highly individual polysaccharides. When
  the conditions of the surroundings arc 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 PNEUhlOCOCCUS

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 pncumococcal 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 pncumococci.
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 diffcr-
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 Specks

The mutability of members of the bacterial tribe Streptococ-
cnceac 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
Pncumococcus to Streptococcus 73iridarks to Strcptococcw hac-
molyticus and back to Pncumococcus. 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.

;
E,

There is no call to recite at any length the accounts of the early
cxpcrimcnts. Some wcrc based on crude, faulty methods which al-

I
h   ways raise doubts as to the purity of the cultures the pioneers

I   studied. Disregarding claims resting solely upon morphological or
B   cultural phenomena, it is better to confine the discussion to re-
r

  ports, with a few exceptions of historical interest, that have been
  published since the development of modern bacteriological and

r..
?   serological technique. In 1891, Kruse and Pansini,7"3 by trans-
t
L   planting forty-six strains of pneumococci on media unfavorable to
E
;i  growth, developed eighty-four varieties that exhibited differences


170       BIOLOGY OF PNEUbIOCOCCUS

in character all the way from typical lliplococcu~s Zanceolatus to
Streptococcus pyogcn~s. 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,
strcptococcal form.

There the matter rested until 1007, when Bucrger and Rytten-
bcrgl" dcscribcd ;~n organism isolated from a case of pucrpcral
pneumococcen~in which, although originally failing to ferment
inulin and cshibiting streptococcal characters, developed into a
typical pncumococcus 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 t,ypical of streptococci which, however, gave way to pneumo-
coccal characters after propagation in mice. Buerger and Rytten-
berg concluded :

Tbc 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 "`* made the stntcment that strains of Strep-
tococcus viridans isolated chicfly 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 pncumococci, and hence designated them as "modified pneumo-
cocci." Roscnow also claimed that during a study of autolysis of
pncumococci 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" description in
1'314 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. zriridans, and
then into a pneumococcus.

Rosenow claimed to have converted into hcmolytic streptococci
eleven strains isolated as pneumococci from sputum, blood, and
the lung in pneumonia and from human cmpyema 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 wcrc said to have been converted into hcmolytic 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 (Marmorck's test), the specific immunological re-
sponse as manifested by the appearance of opsonin and ngglutinin
in antistreptococcic and antipnrumococcic serum, and the more or
less specific pathogenic powers of the various organisms.


172       BIOLOGY OF PNEUhiOCOCCUS

In summary Roscnow 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 prc-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 asccndcncy and tend to persist. This may
hold true in viva 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 cvi-
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 reprcscntcd 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-
cdly 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) )lba4 in a long theoretical discussion of pneumococ-
cal mutation, suggested that the members of the large tribe Strcp-
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 cndocarditis lenta into Pncu-
mococcus. The organism became bile-soluble, optochin-sensitive,
developed a capsule, fcrmcnted inulin, and was Icthal for mice.
Evidence of bacterial mutations of any kind coming solely from in
viva experiments is to be weighed with caution.

Neufeldv70 in a discussion already cited on microbic variability,
recalled an observation he had made ten years previously on the
original "I'neumococcus I" of Neufeld and Haendel, which had
been preserved by drying and storage in a dcssicator. One mouse
inoculated with the culture produced a strain growing in chains,
insoluble in bile, but virulent for mice, and with all typical strcpto-
coccal properties. At first Ncufcld thought he had made a mistake
in the material he injected, but a similar experience of Schicmann's
convinced him that a mutation had actually taken plucc. Coming
from anyone less eminent than Neufeld, this single, isolated obscr-
vation would bc disregarded.

In the following year, Rlorgenroth, Schnitzcr, and RcrgcF' an-
nounced that by special methods they had been able with rcgulnr-
ity to transform pncumococci into streptococci." Their medium
contained dead yeast cells or animal charcoal which had adsorbed
optochin. The altered strains bccamc insoluble in sodium tauro-
cholatc, were avirulcnt for mice, and wcrc resistant to optochin.
Modification A represented the first stage in the transmutation.
The organisms retained the majority of their pncumococcal char-
acters, but were more resistant to optochin and more sensitive to

o Stankal 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. uidans. 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 Strcptococc~m haemolyticus, they produced more or less hc-
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 modifca-
tion C.

Bergcr and Englemann'oo continued similar mutation expcri-
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 fcvcr. 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 tnkc
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 hcmolytic streptococcal colonies on a blood-ngar
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 I'neumococcus after two culture gen-
erations. The original hemolytic streptococci after three culture
generations became green streptococci and after four culture gcn-
erations reverted to pneumococci. This cycle, like Roscnow's, seems
almost too rapid and direct to be credible.

In another communication, Bergcr and Jakob ( 1026)*02 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 lo1 in the next year reported the
mutation of a strain of Type III Pncumococcus through the in-

i  termediary A modification to a green streptococcus. As in their

i  former experiments, the agents necessary for the transformation
  were dry yeast-broth and serum-broth containing one five-thou-
`>   sandth part optochin. Wirtll"Z3 believed that Streptococcus mu-
cosus represented a mutation from Pncumococcus, but he failed in
  his attempts to prove it.

  In yet another paper Bcrger with Silberstein'08 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
I
:  failed to display any action on inulin. The authors then classed

k  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

g.

behavior toward inulin to some thirty &idans strains. Of the lat-
tcr, five exhibited a marked action on inulin, and four others gave
slightly positive reactions.

Reimann,"27 repeating the experiments of Morgenroth, Schnit-
zer, and Bergcr, claimed, however, t,hat 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 pncumococci derived by Morgcn-
roth's method, moreover, were identical with those of R pncumo-
cocci derived by various other means. When one considers the
atypical action of the Bcrger strains on inulin and the author's
omission of serologica tests, one is inclined to accept Rcimann's
interpretation as the correct one.

Heim and Schlirf,"s3 likewise, were unable to verify the work of


176        BIOLOGY OF PNEUMOCOCCUS

Morgenroth and his collaborators, yet Silberstcin,`28a who quoted
these authors, by the aid of optochin in vitro, claimed to have cx-
pcrienccd no difficulty in carrying a Group IV pncumococcus
through the successive stages of Modification B (green Strcpto-
coccus) to Modification C (virulent hemolytic Streptococcus) and
then from this form to a Type I pneumococcus of low virulence.
Paul'070 wns another to join the newer school which believed that
the gap between pncumococci 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.

Giirander (1930) 542 ulso stated that he had transmuted cultures
of Streptococcus viridams into bacterial forms that in every re-
spect were identical with the type-specific pncumococci of human
origin, except that the strains were not agglutinated by antipneu-
mococcic serum. The defect would seem to be a vital enc. The cul-
tural changes were accomplished by repeated cultivation on blood
agar and by short mouse passages. According to Gijrander, after
the third short (four-hour) mouse passage, hemoIytic 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
blood agar and broth), were retransformed "into a bacterium of
perfect Streptococcus tiridans type."

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

DISSOCIATION AND TRANSPORhXATION      177

1:inally single ccl1 cultures originating in their time from a single cell
of a ~~neumococcus have been examined with regard to the degree of
dissimilarity which such cultures can eventually show. `I'hese experi-
merits gave tllc result tllat two pmxmococcus cultures, obtained from
the same cell, can sllow much greater dissimilarities tllan two cultures
obtained one from a typical Streptococcus viridnns and the other from a
typical Strfptococcus Zanceolntus. . . . Tlius Streptococcus viridans
and ~`~L~~L~~~COCCUS Zanceolatus see~n to bc different forms of tllc same
bacterium, and tile specific aggIutinal)ility, which I-`neumococcus Zan-
ceolntus sllows when grown from tile human body and wllicll has been
taken as a base for the so-called type classification, is only an occa-
sional chrscter.

Summary

Virulent pneumococci of all the known serological types, upon
encountering unfavorable physical, nutritional, or other biochcmi-
cal conditions during growth or storage, undergo marked changes
in virulcncc, in ability to clnborutc capsules, in colony dcvclop-
ment, and in their immunological characters. In studies on the dis-
sociation phcnomcna displayed by pncumococci, a great variety
of aberrant coccal forms have been observed which are intermedi-
ate between the typical, virulent form and the thoroughly dc-
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 tbc 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 pncumococci uni-
form with that employed in naming the variants occurring in the
case of other bacterial spccics, it has been proposed to change the
terminology now in USC. Mucoid or M would replace the present
smooth or S ; smooth would be substitutc~tl for rough; while rough
or R would apply to a recently discovered variant. Whatever the
fate of the proposal, the nltcrations 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 antigcnic 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 pncumococcal 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.