129

THE SIGNIFICANCE OF PNEUMOCOCCAL TYPES.

  BY FRED. GRIFFITH, N.B.
(A Medical Oficer of the Ministry of Health.)

, (From the Ministry's Pathological Laboratory.)

COIVTENTS.

I. OBSERVATIOSS ON CLISKAL MATERIAL
   Types in Lobar Pneumonia
   Variety of Types in Sputum from the SFLIJI~ C'nse
   A Rough Virulent Strain
   A Strain agglctinating specifically with two different Group IV Sera
II. ESIWRI~IESTAL NODIFICATIOS
    Attenuation m Culture
    (1) Growth in Zmm~rte Serctm
    (2) Growler on S&d Xedia
    (3) Differences between Indiuidml R and S colonies
   Reversion from Rough to Smooth
    A. Origin of the R Straius used
      B. Passage of R ZZ Strains
      C.  JImsire Dosage with R II
   Inoculation of living R and killed S cultures
    Preliminar~~ Ezperimexf8
    Group I?' S culture + R I and II
    Type Z S culture-t R II and 2
    Type III S culture + R Z and II
    Type II S culture + R I
    Types I uud II S cvlllcres f R Group IV
   Inoculation of living and dead R cultures
III. DISC~SSIOS

IV. SL'MMARY

I. OBSERVAT~OXS ON CLINICAL MATERIAL.

SINCE communicating my report1 on the dist'ribution of pneumococcal types
in a series of 150 cases of lobar pneumonia occurring in the period from April,
1920 to January, 19 22, I have not made any special investigation of this
subject. In the course, however, of other inquiries and of the routine examina-
tion of sputum during the period from the end of January, 1922, to March,
1927, some further data have been accumulated2.
Table I gives the results in two series and, for compariscn,those previously
published.

' Reports on Public Health and Medical Subjects (lW?), So. 13.
' I owe many thanks to Dr J. Bell Ferguson, formerly Medical Officer of Health for Smethwick,
for sending me many specimens from cases of lobar pneumonia.


130         Pnezanzococca~t Types
    Table I. Types of Pseum,ococci in Lobar Pneumonia.
                        Percentage incidence of types
                 Total cases      I              A             7
  Period of inquiry     examined    Type I   Type II   Type III Group IV
Apr. 192OJan. 1922      150       30.6      32.6        6.6      30.0
Feb. 192"Oct. 1924               42.6      21.3        3.2      32.7
Nov. 1974-Mar. 1927   z:       34.3       7.4        4.4      53.7
The main point of interest, since the beginning of the inquiry, is the
progressive diminution in the number of cases of pneumonia attributable to
Type II pneumococcus. The great majority of the cases occurred in the
Smethwick district, and the figures may reveal a real local decrease of Type II,
and a corresponding increase of Group IV cases, It' must, however, be re-
membered that the isolation on a single occasion of a Group IV strain from
a sputum, especially in t,he lat.er stages of the pneumonia, does not prove
t,hat &rain to be t,he came of the disease. This is clearly shown by the
examination of several samples of sputum taken at different times from the
same case; in these a Group IV strain was often found in addition to one or
other of the chief types. There may be a slight element of uncertainty
regarding causal connection of the Group IV strains with the pneumonia,
since the cultures of pneumococci in this series were derived from sputum
(except in four cases where the material was pneumonic lung) and some of
the samples of sputum were obtained when t'he disease had been in progress
for some time-from 4 to 11 days after the onset.

Occurrence of a Variety of Serological Types in the Sputum
     from an individual case of pneumonia.
In my report (1922 Eoc. cit.) I described a number of instances where
several serological varieties of pneumococci were found in the sputum of a
pneumonia patient. One instance was particularly striking, where the sputum,
No. 112, taken on the sixth day of the disease (a crisis had not occurred),
yielded a Type I culture and three strains of Group IV, i.e. four distinct
serological races. On other occasions different specimens of sputum from the
same case, taken at varying periods after the onset of pneumonia, were found
to cont,ain two or more serological types.
Three alternative explanations, at least, are possible.
1. The patient having previously been a carrier of several Group IV strains became
infected with a Type I strain which produced pneumonia. There is no evidence to show
which of the types was present in the pneumonic lung, but I think that the Type I may
be assumed to have caused the disease.

2. The patient when normal wits a naso-pharyngeal carrier of a Group IV strain.
Owing to a cjndition being produced favourable to mutation, a type of pneumococcus, in
this instance Type I, was evolved in his air-passages which was able to set up pneumonia.
On this hypot.hesis, the different serological types would be evidence of the progressive
evolution.

3. On the other hand, the Group IV &rains might be derived from the Type I in the
course of successful resistance against the latter strain. With the increase of immune


F. GRIFFITH

131

substances or tissue resistance the Type I would be finally eliminated, and there would
remain only the Group IV strains which are almost certainly of lower infectivity and
perhaps of less complex antigenic structure.

In the hope of gaining further information on these points I continued
the analysis of the types yielded by the same patient, employing the following
method :

The sputum was preserved in the ice-chest until the preliminary diagnosis
of the infecting pneumococcus had been made in the usual way, viz. by the
intraperitoneal inoculation of a mouse and by testing the peritoneal washings
versus the type sera. The following day some of the sputum was inoculated
together with the type serum corresponding to the strain identified. Fre-
quently the second mouse died from an infection with a pneumococcus of a
different type from that first obtained. If a serum corresponding to the fresh
type was available a third mouse was inoculated together with the sera
appropriate to the two types already identified. The following examples will
make the procedure clear.

(1) A specimen of sputum, No. 239, from a case of pneumonia of four days' duration
was sown on plates, and five colonies of pneumococci were examined, all of which proved
to be Type I; the mouse test also gave Type I. The next day the sputum was inoculated
into a mouse together with Type I serum. The mouse died and the peritoneal washing
reacted only with Pn. 41 (Group IV) serum; the blood of the mouse was plated and of five
colonies examined two were Pn. 41 and three were strains of Group IV which could not
be identified. The sputum was inoculated a third time plus a mixture of Type I and Pn. 41
sera. The third mouse died within 24 hours and its blood yielded a virulent culture of a
Group IV strain which did not react with any of the available agglutinating sera.
(2) A more complete examination was made of the sputum from a second case, No. 273,
of lobar pneumonia, specimens being taken at difIerent periods after the onset of the
disease. The details are given in Table II.

Table II.
Types of pneumococci obtained

Specimen of  Day of
sputum    disease     On direct plate
1        4th     Type I (3 colonies)
           6th     Type I (7 colonies)

   3        8th      Not clone

   4       12th      Kot done
   5       15th
   6       17th    Group IV (1 colcny)

   7       19th   poi `d",;,k" colonu3)

   8       21st      No Pn. colonies

Through mouse

Sputum alone    + Type I serum
Type 1         Pn. 41
Type 1         Pn. 160 and
          Group IV?
TYJ; I=&          -
Type'1             -
Group IV           -
Group IV           -
Type I and          -
  Pn. 160
Type 1             -

The first specimen of sputum collected on the 4th day of the disease yielded pneumo-
cocci of Type I, both on the direct plate and through the mouse. The same specimen of
sputum, which had been kept on ice in the meantime, was inoculated into a mouse together
with a protective dose of Type I serum; the mouse died within 24 hours and a pure culture
of Group IV, viz. Pn. 41, was obtained from the blood. The specimen collected on the
6th day of the disease was examined in the same way and gave a similar result, but on


132

this occasion the protected mouse yielded two different strains of Group IV. One of the
latter was identified with Pn. 160; I had no serum corresponding to the other.

An interesting result was obtained with the 8th day specimen. The mouse inoculated
with t.he sputum died of a mixed infection, and on a plate from the blood it 1va.s possible
to pick out only three pneumococcus colonies. Two of these colonies reacted with PO. 160
serum alone, while the third gave equally good specific clumping (firm masses) with both
Type I and Pn. IGO sera. This third colony culture was plated and the plate showed two
varieties of smooth pneumococcus colonies differing slightly in appearance but easily dis-
tingu&hable. Several of each variety were grown in broth and the agglutination reactions
were tested; rounded dome-shaped pearly colonies were found to be Type I, and larger,
flatter and more translucent colonies were Pn. 160. In a,ddition to the above there were
on the plate a few typical rough pneumococcus colonies, four of which were subcultivated
and tested on mice. Three were avirulent but t,he fourth caused septicaemia in mice and
produced a peritoneal washing which agglutinated specifically with Pn. 160 serum. The
culture obtained from the blood remained rough in cha,racter and thus possessed a com-
bination of rough cultural characteristics and virulence which had not previously been
noted. Further experiments were made with this culture (see p. 11 i).

The above instance may be simply an example of a mixed colony and nothing more.
On the other hand t,here is the possibility that this mixed colony was derived from a
single coccus possessing double antigenic properties. The culture being perhaps in an
unstable condition may have separated in the course of growth into two elements, Type I
and Pn. 160, in each of which the second antigen was suppressed.

On the 12th day the sputum yielded Type I through the mouse. In a specimen taken
three days later, Type I was not found, the direct plate cultures as well as the cultures
through two mice belonging to Group IV but not reacting with any of the available sera.

On the 17th day of the disease there was again no evidence of Type I, each of two
mice being infected with an unknown Group IV strain, while on the direct plate Pn. 160
reappeared. Type I was again found on the 19th day and still persisted in the sputum on
the 21st day after the onset of the pneumonia.

It is curious t,hat the Pn. 41 culture was never again found after the first test. The
strain produced large capsules in t.he blood of the mouse and a peritoneal washing reacted
vigorously with the type serum. The Pn. 160 culture on the other hand appeared frequently
in the course of t'he investigation.

(3) Sputum 218 came from a man who had been ill with pneumonia and had not yet had
a crisis. This specimen yielded Type I colonies both on plates made direct from the sputum
and through the mouse. On the 10th day of the disease, three days aft,er the crisis, a
second specimen of sputum was taken. The sputum alone inoculated into a mouse caused
fatal septicaemia, and plates from the blood gave a pure growth of pneumococci; three
colonies belonged to Type I and seven to an unidentified strain of Group IV. Inoculated
plus Type I serum t,his 10th day sputum killed a mouse and ten plate colonies from the
blood belonged to an unidentified Group IV strain.

On t,he 14th day of the pneumonia the sputum alone killed a mouse, and four,colonies
on the plate from the blood were identified as Type I. Sputum together with Type I
serum yielded through the mouse an apparent'ly pure culture of Type II B (12 plate colonies
were identified). The sputum which had been preserved in the ice-chest was then hocu-
lated into a mouse together with a mixture of Types I and II B sera; this mouse died of
a pure Type III infection.

A final specimen of sputum taken on the 16th day of disease was inoculated into three
mice; (I) with sputum alone and (2) sputum plus Type I serum both yielded Type IIB
only, (3) sputum plus a mixture of Types I and II B sera yielded an unidentified strain
of Group IV,

(4) Six other cases of pneumonia were inveshigsted in a similar manner to the above.


F. GRIFFITH

133

Type I pneumococci were grown from each case and, in addition, Group IV strains were
obtained from five and a Type III strain from one.

(5) A few cases only of lobar pneumonia due to Type II pneumococci have been studied
in the above manner. Sputum No. 267, which killed a mouse with a Type II infection,
was re-inoculated plus Type II serum and the mouse died of a Type III infection.

(6) The lung from a fatal case of pneumonia, No. 230, was plated directly and 34
colonies were examined, all of which proved to be Type II; 12 colonies from a mouse
inoculated with the lung were also Type II.

I have not had an opportunity of ascertaining whether more than one
type of pneumococcus can be obtained from the lung in a fatal case of
pneumonia due to Type I; only in sputum has a mixture of several types
been demonstrated. This latter fact might suggest that the secondary strains,
viz. Group IV and Type III, were present in the upper air passages prior to
the infection with the more invasive strains of Types I and II. On a balance
of probabilities interchangeability of type seems a no more unlikely hypo-
thesis than multiple infection with four or five different and unalterable
serological varieties of pneumococci. Moreover, failure to find more than
one type in the lung of a fatal case of pneumonia would not be conclusive
evidence against t,he modification hypothesis, since the fatal termination
would in itself indicate an absence of those protective antibodies which may
be necessary to initiate an alteration in the type of the infecting pneumo-
coccus. Lung puncture in a case of resolving pneumonia might furnish more
precise indications.

The above findings, taken alone, are not decisive in favour of either of
the two hypotheses, but they assume greater significance when considered
together with the laboratory experiments on alteration of type described later.

Further remarks on a Pneumococcus Strain from Sputum
   producing rough colonies yet virulent for mice.
A distinguishing feature of an avirulent pneumococcus is the rough
appearance of the colonies after 24 hours' growth on a blood agar plate.
Until the appearance of the strain already referred to on p. 116 the above
morphological character of a pneumococcus colony has been found invariably
associated with absence of virulence. The strain in question produced very
typical rough colonies, but nevertheless was able to multiply in the mouse
and cause fatal septicaemia. The blood of the mouse showed pneumococci
with well marked cap&es, and on plate cultures rough colonies grew, identical
in appearance, except in one instance, with those of the original strain.
There are some points of interest both in regard to the origin of the strain
and in the experiments which proved it to combine roughness of colony with
viruIence for mice.

The strain was derived from the sputum, Ko. 273, of a case of lobar pneumonia. Severe1
specimens of sputum from this case were examined at different stages of the disease and
the results are given on p. 116. The sputum which yielded this strain was the third speoimen
and was taken on the 8th day after the onset of pneumonia; it produced ~~~UIUOCOO~


scpticaemia in a mouse and a smooth colony was subcultivated in broth from the blood.
The broth culture on being tested against all the available pneumococcus agglutinating
sera gave a positive reaction with two sera, viz. Type I and Group IV (Pn. 160); in both
cases the coarse masses characteristic of a reaction with soluble substance were formed.
This unusual occurrence was investigated in the folloxing ways.
The original colony culture in broth was plated and on the plate three different varieties
of colonies were identified. Two varieties were smooth, one of which was found to agglu-
tinate with Type I serum and the other with Group IV (Pn. 160) serum; t,he third variety
was rough. Four of the rough colonies were subcultivated and each was inocula.ted subcu-
taneously into a mouse in a dose of 0.25 C.C. of broth culture. Three of the mice were well
when killed three days later and cultures were grown from the seat of inoculation; each
culture thus obtained wvas inoculated intraperitoneally into a second mouse without causing
any ill effects. The mouse inoculated with the fourth colony died of pneumococcal septi-    .
caemia. The blood yielded colonies which were wholly of the rough variet,y and which,
nevertheless, when grown in broth agglutinat,ed typically with Pn. 160 serum.
The broth culture of the original colony was also inoculated intraperitoneally in a dose
of 0.01 CC. into three mice which had received preliminary treatment with protective sera.
The mouse immunised with Type I serum died 6 da.ys after inoculat,ion and a pure culture
of Pn. 160 was obtained from the blood; the mouse protected with Pn. 160 serum died
within 24 hours of a Type I infection; the third mouse treat.ed with the two sera, Type I
and Pn. 160, survived the culture inoculation. The colonies of the two strains obtained as
above were smooth; the rough Pn. 1GO element was elimina.ted.
A series of passage experiments, the details of which are given in Table III, was made
wit,h the rough virulent culture of Pn. 160 to ascertain whether a change into the smooth
form might be induced.

      Table III.
   Rough colony (sputunl 273)
       M. b24
       D. 2 days
         I
        Broth culture
0.001 C.C.     0.01 C.C.      0.1 C.C.

JI. \38   M. 439         I
                    11. 740
D. 10 days    D. 1 day
     Subcut. I      D. 1 day
               Subcut.

10-e
I

    nr. :I30   ?a :62
      K. 2 days    K. 1 day
     Rough culture Rough-smooth cult,ures
                I
              Rougb culture
10-4        10-a        10-Z       0.1 cc.
I           I           I          I

31. $7 1     nr. `ii2    31. b73    fir. b85     M.`786
D. 4 days    D. 26 days   D. 21 days    Survived    D. 1 day
Rough culture  Rough culture Rough culture
                          !            Rough culture
                                              I

subL:cut.
  II. 853
n. 2 days
Rough culture

10-a        10-Z        10-Z

I           I           I

hl. $92    M. `793    nf.' 794
D. 2 days   D. 2 days    D. 2 days
Rough culture Rough culture Rough culture


---

F. GRIFFITH

Throughout a long series of passage experiments, of which those given in Table III
are about half the total, the majority of the mice died of pneumococcal septicaemia and
showed capsulated diplococci in the blood; the cultures obtained from the blood retained
the original rough appearance of colonies with a single exception. The survival of some of
the mice for prolonged periods, up to 26 days, is noteworthy; in the end such mice suc-
cumbed to pneumococcal septicaemia and yielded rough colonies from the blood. All the
rough cultures in broth agglutinated with Pn. 1GO serum.

At one stage of the passage experiment, as mentioned above, a change from rough to
smooth occurred and this ras the only instance observed, although plate cultures had
been made from every mouse in the series. The change from the rather large coherent
colony into a much smaller shiny colony of almost watery consistency was very striking.

The circumstances of the conversion have some interest. The mouse, No. 7G2. from
which the smooth variety was obtained, had been inoculated subcutaneously with 0.23 C.C.
of broth culture of a rough passage strain and was killed the next day. The blood of the
mouse yielded numerous colonies of the usual rough character, among which was detected
one with a small smooth segment. The latter was touched with a fine needle and from it
a second plate culture was made on which grew a mixture of rough and smooth colonies.
From one of the latter a pure smooth colony culture was obtained and this a,a,olut.inated
like the original rough colony with Pn. 160 serum.

The virulence of the smooth colony resembled that of the rough colony and after
passage through four mice in series the same chronic infection occurred ending in death
from septicaemia; for example, one mouse which received IO-5 dose of the smooth broth
culture died 25 days after inoculation. At the end of the passage the smooth colony still
retained its small size, being definitely smaller than the normal Group IV colony.
Both the above cultures, the rough and the smooth, were grown in the antiserum of
t,heir type. From the serum cultures rough and smooth strains were obtained and it waa
found that each variety had become attenuated. Neither would kill mice in intraperitoneal
doses ranging from 0.1 C.C. to 0.2 u.c., the mice being kept under observation for two
months.

I record the foregoing observations without attempting at present to interpret them.

A Strain agglutinating specifically with Sera of two
       Different l'ypes.

The strains of Group IV comprise many different types which are re-
markably well defined and exhibit no cross-agglut,ination amongst them-
selves, provided one takes as the criterion of type agglutination the formation
of firm clumps, either with cultures or with peritoneal exudates. This speci-
ficity is no doubt due to the secretion of soluble substances peculiar to each
type. The strain which I am about to describe gives with two different type
sera the firm clumps characteristic of the reaction between soluble substance
and agglutinin.

The source of the culture was the lung of a woman who died 9 days after the onset
of broncho-pneumonia, A plate made directly from the lung showed large smooth pneumo-
coccus colonies. Seven colonies were subcultured separately and were inoculated intra-
peritoneally into mice, which died within 24 hours. The peritoneal washings from these
mice were found to react with two different Group IV sera, viz. II B and Pn. 87; the
reactions were equal with six of the washings, while the seventh gave a heavier precipitate
with II B serum than with Pn. 87 serum.


136

Pmmxococcal Types

Comparative tests were made with the new strain and with the stock II B and Pn. 87
strains.
         Strain                    11 B SWUIII    Pn.87 Serum
        IIB:    peritoneal washing         -I
        Pn. 87 :     1:                          +
        New strain :                      +           +
The culture of II B agglutinated up to 1 in 320 with II B serum and not at al! with
Pn. 87 serum. Pn. 87 culture agglutinated to 1 in 160 with Pn. 87 serum and gave a trace
of 1 in 20 with II B serum. The new strain in culture agglut,inated with both sera but,
unlike the two homologous strains, did not form firm masses characteristic of virulent
pneumococci. Instead there was produced a turbidity made up of fine granules, showing
that there was probably a deficiency of soluble substance. In the peritoneal cavity of the
mouse, on the other hand, soluble substance is more readily produced and in consequence
the washings gave with both sera the typical react'ion of a virulent strain.
It may be remarked that such behaviour has been observed on a few other occasions;
a pneumococcus strain in culture, obtained direct from sputum, has not reacted with the
type serum while the same strain in the peritoneal exudate of a mouse has reacted typically.
Such a result is no doubt an indication of reduced virulence with associated deficiency in
the production of soluble substance.

A series of experiments were made to prove that the new strain contained two antigens
and was not merely a mixture of II B and Pn. 87. A preliminary test showed that II B
and Pn. 87 sera each protected mice against 04001 C.C. of the new strain but not against
O@Ol C.C. Plate cultures were made from the blood of (1) a mouse injected with II B serum,
and (2) a mouse injected with Pn. 87 serum along with the new strain, and four cblonies
from each were studied. Seven of the colony cultures reacted equally well with both sera;
one gave a stronger reaction with II B serum than with Pn. 87 serum. Cultures were made
in II B serum and were inoculated into mice treated with II B serum; from the mice which
died strains with the double antigens were recovered, whereas if the new strain had been
a simple mixture one would have expected the II B constituent to be eliminated. In point
of fact the II B antigen was probably the major antigen, since some single colony straina
were obtained which reacted only slightly with Pn. 87 serum.

I have recorded my observations on this strain rather fully since it is the
only exception I have found to the rule that a pneumococcus has only one
well-developed antigen. The observation may however be significant as
indicating that this rule is not absolute and that the purity of the specific
antigen in virulent pneumococci may only be apparent.

II. EXPERIMENTAL MODIFICATION.

Production of Attenuated Strains of Pneumococci.

(1) By growth in immune serum.

Culture in homologous immune serum is perhaps the most convenient
method of producing attenuated strains of pneumococci, recognisable by the
morphological appearances of the colonies and termed the R form of the
pneumococcus. Complete attenuation of a virulent pneumococcus culture is
secured only after several passages in series, the first and second serum
cultures generally being composed of a mixture of S and R forms while either
the third or fourth cultures may contain purely R forms. During my fYirst


F. GRIFFITH

137

observations? on this matter I found that the more stable R forms were
obtained from the later cultures in series and I concluded that this was the
effect of the several passages in serum.

As, however, very stable R forms may be isolated from a first serum
culture, and, on the other hand, unstable R strains, those which readily regain
virulence in the mouse, may retain this property after repeated passages in
immune serum, my first experience may have been a matter of chance. It
appears, as the tests on p. 123 show, that R colonies on a plate from an
immune serum culture are not equally attenuated, the capacity to revert to
the smooth type on inoculation into the mouse being more pronounced in
some colonies than in others. Thus it is true that several passages in series
are required to eliminate the smooth form but it cannot be predicted what
the effect of repeated exposure to the action of immune serum may be once
the R state has been reached.

(2) By growth on sol&l media.

Attenuated R strains can also be obtained from virulent cultures by
growth on chocolate blood agar. A virulent pneumococcus culture in blood
broth, plated on this medium and examined after 24 hours' incubation at
37" C., yields as a rule only smooth colonies. If the plate is left in the incu-
bator for two days, some of the smooth discs, which after the first night's
incubation were perfectly regular in outline, develop small rough patches in
their margins. The rough patches may develop into a wedge with the base
at the periphery and the apex at the centre of the colony. They may be
either raised above or depressed below the level of the original smooth growth,
and generally they project beyond its margin. Sometimes the rough area
forms a rounded project,ion extending well beyond the margin of the S colony
and sending a process like a single root towards the centre. Usually a colony
shows only a single rough focus but cultures are variable in this respect,
and some produce colonies which become studded with rough areas. But
many cultures, especially highly virulent strains of Type II, fail entirely to
produce colonies with rough patches.

Rough foci have never, in my experience, become visible in smooth colonies after a
single night's incubation; it is essential that the culture medium should be sufficiently
favourable to allow growth to continue for at least 48 hours. The following is an instance,
The stock virulent strains of Types I and II w-ere plated and produced completely smooth
colonies after 48 hours' incubation. After three days incubation Type I colonies showed
occasional small rough areas but none was seen in the Type II colonies even on the 4th day.
One of the Type II colonies was subcultured in blood broth and then plated; the majority
of the colonies produced were smooth but there were also a few R colonies.
Apparently a few R pneumococci are formed in a culture which is allowed to age on
blood agar and these may multiply and produce a rough area or colony in and perhaps at
the expense of the smooth growth.

When the patch is large it may be touched with the point of a spatula and a pure
     1 Reports on Public Health and Nedical Subjects (1923), No. 18.


138         P~zeiwzococcal Type.3
rough strain may be obtained. Generally, however, when a rough patch is subcultured in
blood broth and plated a mixture of R and S colonies grows.

R cult,ures from rough areas are, so far as I have ascertained, idemical with those
obtained by growth in immune serum.

The tendency to produce colonies &h rough patches seems to be inherent in some
strains and may perhaps indicate deterioration in virulence. It is not removed by a single
animal passage; a Type I strain of medium virulence which produced colonies with many
rough patches was inoculated into a mouse and caused fatal septicaemia. A culture from
the blood was plated and produced a pure growth of smooth colonies after 24 hours' incu-
bation; many of the latter developed rough patches after a second night's incubation.

Rough patches in colonies have been produced only on the chocolate blood medium
which contains fresh horse serum and every batch of this medium ha.s not been equally
favourable. It is possible that the rough change may be due to the presence of immune
bodies in the horse serum. On agar plates without blood pneumococcus colonies quickly
lyse and become almost invisible. If such plates are left in the incubator daughter colonies
may grow out from the lysed colonies, but when these are subcultivated and plated they
almost invariably produce smooth colonies only.

The formation of individual R pneumococci in a smooth culture does not apparently
take place when the culture has ceased to grow. This was shown in an experiment made
to test the viability of pneumococci on ordinary agar.

Fourteen cultures of pneumococci, each of a different serological type, on nutrient agar
slopes were incubated at 37'C. in tubes sealed with paraffined plugs. In 24 hours the
growth had become almost invisible from lysis. The tubes were left undisturbed in the
incubator for two months when they were scraped and subcultivated in blood broth. All
the cultures were viable and the colonies grown on blood a,aar plates were smooth. After
5;1 months' incubation twelve of the cultures were still alive, and on plates, while most of
the colonies produced were smooth; occasional rough ones were detected. After 15 months
in the incubator four cultures still survived; two were completely rough and had lost their
virulence for mice, while the other two produced a mixture of R and S colonies. It will
be observed that the surviving pneumococci remained in their original smooth condition
in the tubes which had not been disturbed for two months. At the end of that period I
suggest that the scraping of the surface and the transference of pneumococci to fresh parts
of the medium caused further growt.h, with the result that R forms appeared. For the same
reason t.he change to the R state was still more advanced at the conclusion of the experiment.

Similarly, as the pneumococci do not grow, no attenuation occurs when the spleens of
mice which have died of pneumococcal septicaemia are dried and preserved for prolonged
periods. I have recovered strains from dried spleens after 34 years a.nd have found the
virulence unaltered. The surviving pneumococci may be very few in number and may be
recovered in the following way. The whole of the spleen is ground to a fine po-wder in a
mortar and emulsified in a small quantity of blood broth. This may be plated directly or
after a few hours' incubation. The colonies produced have always been of the smooth form;
the pneumococci have remained dormant and there has, therefore, been no opportunity for
the production of the R forms.

     (3) Diferences between individual R and S colonies.
Virulent pneumococci which have been grown in homologous immune
serum and have undergone the change from the S to t,he R form are not all
equally affected. Pure R colony cultures show differences amongst them-
selves in (1) capacity to revert, (2) type of agglutination and (3) immunising
properties.


F. GRIFFITH               139

A virulent Type I strain was grown in Type I serum for two generations,
the second of which was plated. Six R colonies were taken and grown in
small quantities of blood broth; these colonies were identical in appearance
and gave non-specific agglutination in pneumococcal type sera. Each colony
culture was subcultivated in 10 C.C. of broth; this was centrifuged and the
deposits were inoculated into mice subcutaneously.

1st inoculation experiments.

Mouse

Result

1         923         Died 2 days.  S colonies from blood
2         924              ,J         ,,       ,I

3         925
4         926
5         927

Survi;kd 12 days. `hiI in blodh
Died 4 days. S colonies from blood

         6         928       Survived -
Each of the six colony cultures was then plated and an isolated colony was
grown which was again inoculated subcutaneously in a dose of 10 C.C. of broth
culture deposit.

2nd inoculation experiments.

Mouse

Result

         1         967      Died 2 days. 8 coloniw from blood
         2         968             ,9         9,       I,
        3         969                      I,       I>
   ii         970      Survi;kd
                  971      Died 3 days. S colonies from blood
         6         972        Survived
In order to make certain that the colony cultures were free from any
S forms of pneumococci, each culture was again plated and an isolated R
colony was grown in blood broth. This procedure (plat,ing and selection of
colonies) was carried out six times in succession. Broth cultures were made
from colonies on the final plates and these were inoculated subcutaneously
into mice in the same doses as before.

3ra inoculation experiments.

Mouse                  Result

Died 2 days. Pneumococcal septi-
caemia, culture overgrown
Died 2 days. S colonies from blood
g;tl;$ddays. ,, ,,
Died 2 days. S colonies from blood
Survived

The results were practically identical in each of the three series of inocu-
lation experiments; four of the R strains reverted in the mouse to the smooth
type, while two, Nos. 4 and 6, were more completely attenuated. The different
degrees of virulence were retained after seven successive platings, thus showing
that the characters were stable and were the property of the whole strain
in each case.


The following experiment reveals similar ditrerences between individual R colonies.
Four cultures made from different colonies of a rough Type I strain were inoculated
into mice in doses of 0.15 C.C. intraperitoneally. Cultures 1 and 2 killed t,he mice in two
da,ys and S colonies were grown from the blood. The mice inoculated with cultures 3 and 4
survived. The latter two mice were reinoculated on the 6th day with 0.25 C.C. and on
the 13th day with 0.5 c.c., each with the same living broth culture as before. They were
tested on the 26th day after the first inoculation with IO-6 C.C. of a virulent Type I culture.
Both mice died, thus showing absence of immunity.
The immunising capacity of the other two rough cultures, 1 and 2, was tested in the
following way.
Xo. 1 was inoculated intravenously into t.hree mice, each with 0.2 C.C. of iiving broth
culture. They resisted this inoculation ond a test dose of virulent culture was given 18 days
later. All of these survived. A similar intravenous experiment with No. 2 failed to produce
immunity against the same test dose. No. 2 was then used to vaccinate 3 mice, inocu-
lated intraperitoneally twice at 7 days' interval. Tested 21 days after the fist vaccinating
dose, two out of these mice survived.
Thus two of the rough cultures produced immunity and two failed. The properties of
the four strains are given in the following table:
   No. of rough   Reversible to    Immunising
      culture     S type       capacity       Agglutinability
        1         Yes          Positive       Type specific
   f      G     Ne&ive     Non-s'iecific
        4          ,,             ,,                7,
In this instance by type specific in regard to agglutinsbility is meant that though agglu-
tination was of the rough character, i.e. the deposit was readily shaken up, it occurred
up to the full titre 1 in 160 of a smooth Type I serum. The non-specific cultures agglu-
tinated to 1 in 10 only with both Types I and II sera.
An experiment on similar lines was made with a rough Type II culture.
Six rough strains from individual colonies were inoculated into mice in doses of 0.75 C.C.
and 0.1 C.C. of broth culture intraperitoneally. All the mice which received the larger dose
died within 24 hours, but in no case was there any reversion to the smooth type. The
peritoneal washings were tested against Type II serum and several gave a slight precipitate
showing probably the formation of a small amount of soluble substance; two gave no
precipitate. The mice injected with 0.1 C.C. all remained well; they were reinoculated after
6 days with 0.2 C.C. and after a further 7 days with O-3 C.C. of the same cultures as before.
The immunity of five mice (one died accidentally) wss tested 26 days after the first in-
jection with lOme c.e. of a virulent Type II culture. Three mice resisted and two succumbed.
It is interesting that the two mice which died were immunised with the two cultures
which failed to produce a trace of soluble substance in the peritoneal washings. The change
to the R form is apparently less complete in some pneumococci than in others, and it is
possible that the retention of a small amount of the original S antigen in their composition
may explain the ca,pability of certain R st,rains to immunise against a virulent pneumo-
coccus as well as their tendency to revert to the S form.
On the other hand, while diminution of virulence in a pneumococcus
culture may be due to a proportion of t,he individual organisms composing it
having undergone the change into the R form, this is not invariably the
case. Cultures which produce only smooth colonies may possess intermediate
grades of virulence, killing mice in doses not less than 0.1 C.C. or 0.01 C.C. of
broth culture. This has been observed in cultures immediately after reversion
from the rough to the smooth form. In such instances the change from the


               F. GRIFFITH                141
one form of antigen to the other may not have been complete, and thus
larger numbers of organisms are required to produce a sufficient concen-
tration of those substances which neutralise the protective fluids of the animal
and enable the bacteria to multiply.

     Reversion from Rough to Smooth.
   A. O,rigin of the rough strains used; eflect of di$erent sew.
The rough Type II strain was obtained in the following way. A virulent

culture of Type II, which killed mice when inoculated intraperitoneally in a
dose of lo-* c.c., was sown into undiluted Type II serum and was passed
from serum to serum for six generations, each of which was incubated at
37" C. overnight. The final culture was plated, and five rough colonies were
selected and subcultured.

A test was made to show that these five subcultures were free from smooth
virulent pneumococci; the procedure being the same for each, a single descrip-
tion is applicable to all. Each rough culture was plated and an isolated
colony was grown in blood broth. A mouse was inoculated intraperitoneally
from the blood broth culture and a plate was made. This procedure, plating
followed by selection of rough colony and mouse test, was repeated six times
in succession. In no case did any of the plates show a smooth colony, and
all the mice (a total of 30) survived the intraperitoneal inoculation of culture,
the doses of which ranged from 0.1 C.C. to 1.0 cc. The final cultures were
tested against agglutinating sera of Types I and II wit'h both of which only
minute clumps were formed, thus showing that the type characteristics had
been lost.

A virulent culture of Type I was treated as above, except that the trans-
ferences were limited to five. The final rough cult,ures were tested on mice
in the same way as those of the Type II. Although the appearance of the
colonies was typically rough, t'hey were found to revert readily in the mouse
to the smooth virulent variety. One culture was then passed through five
more generations of the same batch of Type I serum. The fifth serum culture
was plated and several rough colonies were subcultured and these also were
found to revert readily to the smooth form on being inoculated into mice.

Another protective Type I serum was then taken and a culture was started
with a trace of blood from a mouse which had died from a Type I pneumo-
coccal septicaemia. Four generations of serum cultures were made in suc-
cession, a night's `incubation intervening between each. In the first two
generations the culture grew in the form of a firm mass at the bottom of the
serum; the third culture was partly granular and the fourth was quite diffuse.
Plate cultures were made from the first, third and fourth generations and five
colony cultures from each plate. These cultures were tested on mice, 27 mice
with the first generation, six wit'h the third and six with the fourth, the doses
ranging from 1 cc. of broth culture up to the deposit of 50 C.C.


All the strains, those from the first as well a,s the fourth serum cultures,
were avirulent and none reverted to the smooth form.

It was clear that the second batch of Type I serum used was more eEcient
in producing attenuation than the first. In spite of eleven passages, the latter
did not succeed in removing from the rough strain its ability to revert to the
smo0t.h on inoculation into mice.

A test was made to discover whether growth in the second more potent
serum would further attenuate the readily reverting rough strain. After a
night,`s incubation in the serum a plate was made and three rough colonies
were subcultivated. These were tested on mice in subcutaneous doses of
1 C.C. of blood broth; two of the mice died and smooth colonies were recovered
from the blood; t,he third mouse survived.

Thus the more active serum did not attenuate the rough strain so com-
pletely as it did the virulent capsulated pneumococcus sown from the blood.
It is possible t,hat pneumococci may to some extent become habituated to
t.he action of the serum. For example, treatment with too low a concentra-
tion of protective antibodies seems to have induced the formation of a rough
but reversible strain upon which the serum could no longer act.

The R culture of Type I most frequently used in the subsequent experi-
ments was made from a rough colony (No. 3) grown from the first generation
in the more powerful Type I serum referred to above.

B. Passage experiments through mice.

The following is an example of many similar experiments which I have
made to discover whether an avirulent R pneumococcus can be transformed
into the virulent S form by growth in the body of the mouse. As a rule, the
experiment has been started with the inoculation of one mouse and several
lines of passa.ge have subsequently developed, in only an occasional one of
which has the transformat,ion into the virulent form been effected. This
irregularity of reversion has been a feature of the experiments where the
cult.ure has been passed through a succession of mice in small doses and by
the intraperitoneal method of inoculation. Instances will be given later to
show that a greater regularity may be attained when very large doses, via.
the cent.rifuged deposit of 50 to 100 C.C. of broth culture, are inoculated under
the skin, though even then only a small proportion of the mice succumb to
pneumococcal septicaemia where a thoroughly attenuated strain has been used.

This particular passage experiment was begun with rough Type II strains obtained as
described on p. 125 and was continued along five separate linej. Pure line strains from
single organisms were not used, but the preliminary tests on mice showed that the highly
virulent pneumococcus had been eliminated by growth in the immune serum. As a further
precaution, each of the five strains was plated and an isolated rough colony was grown in
blood broth; this procedure was repeated sis times and colonies from the final plates were
made the starting point of the passage.


F. GRIFFITH               143

One of the five lincq of passage is shown in Table IV.

`Pable IV.

Rough colony culture No. 1.
Deposit of 4 C.C. of broth culture inoculated subcutaneously
     I

11. `690
Killed 2 days
   I

M. 691
Survived

Locall lesion culture
Subcut.

  I
M. 713
Killed 2 days
Locall lesion culture
Rough colony
sulmlt.
DepoTit of S C.C.

I
31. 723
Killer: 2 days
Local'lesion culture
~;p$colonY
Deposit of 100 C.C.
   I

M. A4
Survived

I
iv. 747
Died 3 days
Smooth culture of TII
virulent in a dose of 1O-8

I
AI. 746
Died 6 days
  I
Rough colonies from local lesion
Intrap.
741. 80'1
Killed 9 days
P.M. culture
Nil

The first two mice each received the centrifuged deposit of 4 C.C. of broth culture under
the skin of the inguinal region. One mouse survived; the other was killed after two days,
and from the tissue around the inguinal gland a blood plate and a blood broth culture
were sown. Both cultures were pure, the plate showing rough colonies only, and the blood
broth was inoculated into M. 713 in a dose of 0.5 C.C. Mouse 713 appeared well when killed
two days later and a plate culture was made from the local lesion. A few small rough
colonies grew and one of these was grown in broth.  From this culture two mice were
inoculated, each with the centrifugalised deposit of 8 c.c., under the skin of the inguinal
region. One mouse, No. 723, was killed when well two days later and a plate culture from
the iocal lesion yielded a few rough colonies with one of which the passage was continued.
The fellow mouse was allowed to survive.

M. 723 culture was grown in 100 C.C. of broth and the centrifuged deposit was divided
equally between two mice, both being inoculated subcutaneously. One mouse, NO. 747,
died in 3 days of pneumococcal septicaemia, the blood showing numerous capsulated
diplococci. A smooth culture was obtained from the blood and this killed mice, inoculated
intraperitoneally, in a dose of lo-" C.C. of brot,h culture.
The fellow mouse, No. 746, died six days after inoculation; the blood ww sterile and a
few rough colonies were grown on a plate from the seat of inoculation. One of these colonies
was grown in blood broth and a mouse inoculated intraperitoneally with 0.5 C.C. remained


well. The passage experiments with the other four rough colony cultures ended, as in t,he
example described above, with the subcutaneous inoculation of two mice, each with the
centrifuged deposit of 50 CC. of broth culture. The results are as follows: in No. 2 passage
both mice yielded smooth virulent cultures; Nos. 3 and 4 were like that first described,
one mouse yielding rough colonies and the other smooth; in No. 5 the final cultures obtained
from both mice were still rough and avirulent.

,4n intermediate rough culture in the fifth line of passage was taken and was again
inoculated into two mice. One received subcutaneously the deposit of 66 C.C. of broth
and the other the deposit of 33 C.C. The former died of pneumococcal septicaemia in
three days, the blood yielding a smooth culture; the latter was killed after two days and
rough colonies only were obtained from the local lesion.

In the above experiments it will be noted that the reversion from rough to smooth
occurred in. those mice which were inoculated subcutaneously with large amounts of culture.
It seemed possible that this latter circumstance in affording a favourable nidus may have
had more influence on the development of smooth characteristics than the transference
from mouse to mouse. In order to test this view, four of the original rough cultures were
inoculated subcutaneously into mice, each of which received the deposit of 5Oc.c. Five
survived and were healthy when killed ten days after inoculation; one died in three days and
yielded a few rough colonies; only one died of septicaemia with numerous capsulated
diplococci in the blood, from which a smooth culture of Type II was obtained. It may be
mentioned that none of these cultures reverted when inoculated in a small dose, so it
appears that a large dose favours the reversion, but does not produce it infallibly.

In the following experiment the passage was begun with two R strains of Type II
which had been subcultivated for over a year and had been repeatedly both plated and
tested on mice without producing any S colonies or showing any virulence for mice in
moderate doses. Each culture was inoculated intraperitoneally into a mouse in a dose of
1 C.C. of brot,h culture. The mice were ill when killed the next day snd plat,e cultures from
the blood yielded sparse rough colonies. An R colony strain was made from each mouse
(for convenience of description these have been designated A and B respect,ively); each was
inoculated subcutaneously into three mice in doses of 1 C.C. of blood brot,b. ,411 the mice
survived for three days when they were killed apparently well and plate cultures were
made from the seat of inoculation.

Result of inoculation of rough culture A.

Mouse 1 yielded a mixture of R and S colonies from the seat of inoculation. 9 sub-
culture was made from each variety and tested on mice. The R strain failed to kill in a
dose of 0.5 C.C. intraperitoneally; fatal septicaemia was produced by the S strain in a dose
of 0.1 C.C. intraperitoneally and 0.5 C.C. subcutaneously, but not in smaller doses.
Mice 2 and 3 yielded R colonies only which were not virulent when inoculated into
other mice.

Result of inocz&ztion of rough culture B.

Mouse 1 gave only R colonies on the plate fro-m the local lesion. A culture from one
wae inoculated subcuta,neously in a. dose of 1 C.C. into a mouse which died in twq days from
pneumoccccal septicaemia; typical S colonies of Type II were grown from the blood.
Mouse 2 yielded a majority of R colonies and one S colony which agglutinated with
Type II serum, a.n R colony was avirulent; t,be S colony killed mice in a subcutaneous dose
of 0.1 CC. but not in smaller doses.

Mouse 3 yielded R colonies which after two further passages in series through mice
remained rough and avirulent.

This experiment shows that the ability of an attenuated R strain to revert to the
virulent S form persists during prolonged periods of subculture in the R form.


--

F. GMFFITH               145

C. Inoculation of large doses of R pneumococci subcutaneously into ,mice.
The following table gives the results of inoculating the deposits of broth
culture of a single strain of attenuated R pneumococcus under the skin

of mice.

Mouse      Culture
144    Rough Type II
745          >.
746          ,P
747          ,,
748          ,T
749          3,
750          ,.
751          7,
752          71
753          1.
759          0
760          1.
761          I,
762          ,>
763         ,,
764          9,

I

   Dose
Deposit of 50 C.C.
  ,Y    ,,  ,.
  3.    ,.  ,,
  ?I    I,  ,,
  ,I    I,  0
  9.    ,   7,
  19    ,,  1,
  ,,     9,  2,
   ,I    ,,  1,
  I,    ,,  I,
  ,>

         Result
Died  3 days. R colonies only
,,    3 ::  S colonies, Type II
,,    6    R colonies only
,,    3  ,,  S color.ies, Type II
K%ed 10 5 ,. ,,  6 colonies, Type II
       R colonies only
Died   4  ,,  S colonies, Type II
g;:;d 13 ,>   Culture-nil
Killed 1; ::  S colonies, Type IT
       R colonies only
Died :! ,,  S colonies, Type IT
Died   2  ,,  S colonies, Type II
Survived
Died  1 day.  S colonies, Type II
K&d 2 6 days. ,, S Cultrwe-nil colonies, Type II

These experiments support the view expressed earlier that the subcu-
taneous inoculation of a mass of culture under the skin furnishes a nidus in
which the R pneumococcus is able to develop into the virulent capsulated
form and thence invade the blood stream. As mentioned earlier, passage
experiment.s with smaller doses up to 1 C.C. of broth culture have also resulted
in reversion to the virulent S form, but the occurrence is infrequent and
irregular. The effect of the larger doses inoculated subcutaneously is more
certain and in only 7 out of the 16 examples given has the R strain remained
unaltered.

Inoculation of Attenuated It yneumococci together with
       virulent S culture killed by heat.
           Preliminary Experiments.
The development of the virulent S form of pneumococcus from the R form
inoculated in large doses under the skin of the mouse is no doubt favoured
by the mass of culture forming a nidus. This protection from the normal
defensive mechanism of the animal tissues cannot, however, be the sole
factor in the production of the change, since the attenuated R pneumococcus
may survive unaltered in the subcutaneous tissues for two or three weeks
without any such protection.

Some R strains revert to the virulent type much more readily than others,
and it is possible, as I have suggested above, that such strains may have
retained in their structure R remnant of the original S antigen insufficient in
ordinary circumstances to enable them to exert a pathogenic effect in the
animal body. When a strain of this character is inoculated in a considerabIe
mass under the skin, the majority of the cocci break up and the liberated


S antigen may furnish a pabulum which the viable R pneumococci can
utilise to build up their rudimentary S structure. The a,mount of S ant,igen
in an R strain, even one only partially attenuated, might not be very large,
and it might happen that such an R strain did not liberate in sufficient
concentration the stimulating or nutrient subst,ances necessary to produce
reversion. It appeared possible that suitable conditions could be arranged
if the mass of culture was derived from killed virulent pneumococci, while
the living R culture was reduced to an amount which, unaided, was invariably
ineffective. There would be thus provided a nidus and a high concentration
of S antigen to serve as a stimulus or a food, as the case may be.
A number of experiments on the above lines have been done and the
results have shown quite conclusively that reversion of an attenuated R
pneumococcus to its virulent prototype can be induced with much greater
regularity tha.n after inoculation of large amounts of the living R st,rain alone.
The details of the procedure are given in the following example. A virulent
culture of Type II was grown for a few hours in glucose broth, a fairly dense
growth being produced, and was then killed by steaming at 100" C. The        )
culture was concentrated by centrifuging a,nd four mice were inoculated
subcutaneously each with the deposit of 50 C.C. together with O-5 C.C. of a
living serum brot'h culture of R Type II. All four mice died in 3 to 5 days
with numerous capsulated diplococci in the blood, cultures from which gave
the typical agglutination reaction of a virulent Type II strain. A control
experiment was made at the same time.  Ten mice were inoculated subcu-
taneously each with the same amount of the living R stra.in as above together
with the steamed deposit of 40 C.C. of broth culture of Type I. One of the
mice died 2 days later from an infection with Gram-negative bacilli; the rest
were killed healthy in 7-10 days.  Cultures made from the tissues at t,he
seat of inoculation remained sterile except in two mice, killed after 7 days,
which yielded a few R colonies of pneumococci. This control experiment shows
(1) that the R pneumococcus of Type II remained attenuated in t,he
   absence of steamed virulent Type II, and
(2) that it was not assisted to regain virulence by the presence of t,he
   steamed Type I culture.
The reverse of the above experiment was then tried, the attenuated
culture being the R form of Type I, but in this case there was found a
difference in the results according to the temperature to which the heated
virulent culture was raised. In an experiment with four mice steamed Type I
(deposit of 70 C.C. of broth culture) together with 0.25 C.C. of the R cultures
had no effect, all the mice being perfectly healthy when killed 17 days later.
The same amount of Type I culture heated to 80" C. for one hour caused one
mouse to die 10 days later and a virulent Type I culture was recovered from
the blood; the other three mice were killed after I7 days and cultures from
the seat of inoculation showed t'hat the R pneumococci had perished.
The effect of Type I culture heated at 60" C. for two hours was then tried.


               F. GRIFFITH               147
Six mice received subcutaneously the deposit of 60 C.C. of heated culture
together with 0.25 C.C. of the rough Type I culture; five out of the six died of
Type I pneumococcal septicaemia. In a repetition of the last experiment the
same R culture of Type I in a dose of 0.25 C.G. was administered together
with the deposit of 110 C.C. of virulent Type I culture which had been heated
for two hours at 60" C. Ten mice were inoculated subcutaneously; one died
within a few hours; the rest succumbed to pneumococcal septicaemia in
2-6 days and smooth Type I cultures were grown from the blood in every case.
A control experiment with the above rough Type I, inoculated plus the
deposit of 100 C.C. of virulent Type II culture which had been steamed for
15 minutes, was negative. Ten mice were used and all were healthy when
killed after lo-13 days. Th; R strain had disappeared except in one mouse
where a few R colonies grew from the seat of inoculation.
In t,he preceding experiments the steamed Type II antigen exerted a
specific influence since it caused only the R form of Type II to increase in
virulence and had no effect on that of Type I. On the other hand, virulent
Type I antigen appears to be injured by steaming at 100" C. and produces
no effect either on the corresponding R form or upon the R form derived
from Type II.
If the virulent Type I culture is heat,ed only to 60" C. it very readily
changes the R form of Type I into the virulent S form. But the lowering
of the temperature at which the virulent culture is killed has other important
results, as will be shown later. It may be me-ntioned now that the shorter
                           ..xI. ----
the period durin
          . . h-------- ~----~..-  .._.. -.   ._
             0` which the culture is heated to 60" C. (lower temperatures
have not been tried), the more powerful and the less confined to its own
type is the effect of the virulent antigen on the attenuated R form when the
two are-injected simultaneously into mice. For example, virulent cultures
of Type I heated to 60" C. may cause the attenuated R strain of Type II to
assume the capsulated S form in the animal body.
Short exposures at 60" C. introduce a risk which can be excluded when
the cultures are killed by steaming at 100" C., the risk namely that some
of the pneumococci in the culture may have survived the heating and be still
viable. To ensure that the culture deposits have been uniformly heated,
they have been enclosed in a sealed glass capsule and immersed in a large
water-bath, the whole capsule being kept below the level of the water which
has been carefully maintained at the required temperature. The heated
deposits have been tested by incubation followed by plating and by the in-
jection of large amounts subcutaneously into mice. These tests of viability
have invariably been negative and exposure for so short a period as 15 minutes
to a temperature of 60" C. appears to be sufficient to kill pneumococci. But
the results of inoculating attenuated R strains into mice together with heated
suspensions of virulent cultures of different types have been so remarkable
as to raise the question whether the ordinary tests of viability are sufficiently
comprehensive. It becomes necessary to consider whether heating to 60" C.


148         Pstem~ococcal Types
may produce resistant forms of pneumococci which do not multiply except
when injected into a mouse together with a living attenuated pneumococcus.
On account of the importance of the correct interpretation of the experiments
which I am about to describe, I have given in detail a number of examples
varying slightly in the experimental conditions and in the method of control.
As the heated S culture appears t,o be the determining factor, each series
of experiments is headed by the particular type of virulent pneumococcus
furnishing it. All inoculations designed to increase virulence or alter type
have been done subcutaneously in mice.

Inoculation experiments with heated virulent Group IV and attenuated
        R Type I and R Type II pneumococci.
Table V shows that three different strains of Group IV killed by steam
at 100" C., when injected into mice toget,her with living attenuated R pneumo-
cocci derived from Type II by growth in homologous immune serum, caused
the R form to revert to the virulent capsulated S form. R 4, Type II, i.e.

     Killed S
   pneumococci
Pn. 85, GroupIV, skamed
20 mins. Dose =deposit
of 60 CC. of broth culture

l'n. 160, Group IV, as
above

II B, Group IV, as ahove

Table V.

  Living R
pncumococci
R 4, Type II. Dose
=0.25 cc. of blood
broth culture

R 4, Type II as
above

R 4, Type II as
above

R 4, Type II. Doses
=0.75, 1.0, 1.0 CC.
of blood broth cul-
ture

No. of
mouse

405
406
407
408
409
410
411
412
413
414
415
416
462
463
464

Result

Died  4 days
Killed 7 ,,
D::d :: ::
Killed  7 days
Died   4  ,,
,,
D::d 3 ii ::
    days
I.    2  ,,
Kiiled 7" ::
Killed  19 days
,,   19  ,,
,,   19  ,,

Type of culture
obtained from
   mouse

S colonies, Type II
None
R colonies
S colonies, Type II
S colonies, Type II
   9,       ,,
   7,       9,
S cokirs, Tyljk II
   ,,       ,,
R coibnies "
None

,,
9,

rough colony 4, Type IL, which has been used throughout these experiments
is so much attenuated as never to kill mice in doses of 1-O C.C. of blood broth
culture. The three control mice remained well and were killed 19 days after
inoculation. As mentioned earlier, all the inoculation experiments to increase
virulence have been done subcutaneously and it has been the custom in the
case of mice killed when apparent,ly well to make plate cultures from the
tissues at the seat of inoculation, viz., the right groin. Generally the plates
are either st,erile or purely pneumococcal, but care must be taken should
the inoculated material cause the skin to ulcerate. Extraneous organisms are
usually staphylococci which cause no difficulty. There were addit,ional controls
to the above experiments. These have not been given in the table, but they
show that the stimulus to reversion of the R st,rain was not possessed by


F. GRIFFITH

149

steamed Type III culture (deposit of 60 C.C. for each of 12 mice) or by scar-
latinal streptococci (4 mice).
                   Table VI.
                                     Type of culture
     Killed S         Living R     No. of               obtained from
   pneumococci       pneumococci    mouse    Result          mouse

PI-I. 85, Group IV, heated  R 4, Type II. Dose   791
2 hours at 60' C. Dose =    =0.25 C.C. of blood   792
deposit of 50 cc. of broth   broth culture      793
culture
P,;;,v"e", Group IV, as  R 6, Type I, as   794
                  above          795
                               796
P;boJiO, Group IV, as  R 4, Type II, as 773
                  above           774
                               775
P;bo;zO, Group IV, as Rab:veType I, as   776
                                 777
                               778

Killed 7 days   None
Died   3  ,,    S colonies, Type II
11    3  ,9       ,,       ,,

Killed 16 days
,,   16  ,,
I,   16  .,
Died   2 days
,,    3  9,
9,    2
Killed 15 diys
1,   15  ,,
3.   15  ,,

None
,P

S colonies, Type II
   19       ,,
Non:       ,P

II
11

II B, Group IV, as above  R 4, Type II, as 785  Died 3 days  S colonies, Type II
                  above         786 ,,
                               787    I "       ,*       ,.

II B, Group IV, as above  R 6, Type I, as 788 Kylled 15 diys  Non:       It

                  above         789 I, 15 ,,
                 790 1, 15 I, :;

Table VI confirms the conclusions drawn from the experiments in Table V,
that Group IV S antigen enables attenuated Type II pneumococci to become
virulent in the mouse.

In addition it shows that Group IV antigens, contained in three virulent
strains, after heating to 60" C. for an hour on two occasions, did not produce
reversion of the R form of Type I; the mice inoculated remained well and
no culture could be obtained from the seat of inoculation 15-16 days later.
The latter part of the experiment, which shows that an attenuated R
cult,ure of Type I is not increased in virulence through the influence of heated
S antigens of Group IV, was repeated with identical results. The same three
Group IV strains, heated at 60" C., were injected in doses of 100 C.C. of broth
culture deposit together with a different rough culture, R 3, Type I (this
signifies rough colony 3, derived from Type I and this strain has been most
frequently employed in these experiments). All the mice, a total of 21, were
healthy when killed 22-28 days later.

These experiment,s may be summarised as follows.
Group IV virulent pneumococci, killed either by heating to 60" C., or by
steam at 100" C.: when injected in large quantities of culture deposit into
mice together with an attenuated R strain of Type II caused the latter to
revert to the capsulated virulent form and set up fatal septicaemia in the mice.
Under similar conditions three Group IV cultures failed to increase the
virulence of attenuated R strains derived from Type I.


150         Pnemaococcd Types
Inoculation experiments with heated virulent Type I culture and attenuated
         R strains of Types I and II.
Conversion of R Type II ido S Type I. In the experiment in Table VII
two out of eight mice injected with heated virulent Type I culture together
with an attenuated R culture derived from Type II died of pneumococcal
septicaemia and yielded pure S colonies of Type I from the blood; plates from
the lesions at the seat of inoculation showed a mixture of R and S colonies.

    Killed S
  pneumococci
Type I heated 3 hours at
60" C. Dose = deposit of
50 cc. of brot,h culture

As above

As above

Table VII.

  Living R     No. of
pneumococci    mouse
None            641
,t             642
I,             643
R'k, Tvpe II. Dose   644
              645
=0.25 C.C. of blood   646
broth culture
              647
               648
R 4, Type II, grown   649
in the heated Type
I deposit. Dose =   650
0.36 C.C.         651
              652

  Result
Killed E days
9,      ,,
,P
Dizd : 3 ::
    days
Killed  5  ,,

.,

,f  : ::
Killed  5 days

Died   4  ,,
Killed  6  ,,
9,   6  ,f

Type of culture
obtained from
    mouse
None

,1
I,
S `colonies Type I
R ~01s. f&m local
lesion

   I,       3,
R ck. from ;bcal
lesion
S colonies, Type I
None
One R colony

The remaining six mice were killed, apparently healthy, after 5-6 days and
plate cultures made from the subcutaneous seat of inoculation either pro-
duced only R colonies or remained sterile.

The four mice which received the heated Type I culture alone were well
when killed 5-6 days later, and plates made from the subcutaneous tissues
at the seat of injection remained sterile.

The rough attenuated Type II culture has apparently in two instances
been changed into a virulent Type I. The most obvious alternative to this
presumption is that a few Type I pneumococci in the heated culture remained
viable. It is therefore desirable to give in detail the cultural tests and the
treatment to which the virulent culture had been submitted. After heating
for one hour at 60" C. a large loopful of suspension was sown on blood agar
plates. The tube containing the thick suspension (the deposit of 600 cc.
concentrated by centrifuging to 12 c.c.) was resealed and again heated to
60" C. for one hour. The suspension was then incubated at 37" C. overnight
and plated the next day. All the above plates remained sterile. In order to
show that the thick suspension would not inhibit the growth of viable pneumo-
cocci 4 C.C. of it were sown with the R strain of Type II, incubated overnight
and plated; a profuse growt,h of R colonies only was obtained. This culture
in the suspension was used for the last four mice in the table.

Jn view of these precaut,ions, I think it may be assumed that the heated
suspension contained no viable Type I pneumococci. The R strain of Type II,
although not tested alone on this occasion, has been used throughout these


F. GRIFFITH               151

experiments and numerous tests have shown that it is a pure attenuated
rough culture.

A second experiment (see Table VIII) on the same lines was made and the
cultural tests of viability of the heated culture were as rigid as possible. The
thick suspension of the concentrated broth culture after heating was distri-
buted in ten small tubes so that each tube contained the deposit of 100 C.C.
and to each of them was added 1 G.C. of sterile bovine serum. The mixtures
were incubated overnight and subcultures were made the next day both upon
fresh blood agar plates and in blood broth; the latter were also plated after
incubation at 37" C. The cultures from every tube remained sterile.

Table VIII.

                                   Type of culture
     Killed S         Living R     No. of              obtained from
   pneumococci       pneumococci    mice     Result          mouse
Type I heated 3 hours at   None             4   Killed 7 days   None
60o C. Dose =deposit of
50 C.C. in salt solution
Type I as above. Dose=   None             2   Killed 7 days   None
deposit of 100 C.C.
Type I as above. Dose =  R4, TypeII. Dose 8   Killed 7 days   R colonies from 4
deposit of 50 C.C.        =0.2 C.C. of blood                 and none from the

                 broth culture                       rest
None           R 4, Type II. Dose    2   Killed 13 days   None
                  =0.5 C.C.
Type I as above. Dose =  R4,TypeII. Grown 2   Killed 13 days   None
deposit of 100 C.C.       in heated culture
Type I deposit of 100 C.C.  R4, TypeII. Dose    1   Died  3 days  S colonies, Type I
(bovine serum not re-   =0.25 C.C.
moved)

Seven of the tubes of suspension were centrifuged; the supernatant serum
was removed and replaced by O-5 C.C. of salt solution. Eight mice were
inoculated subcutaneously each wit.h the deposit of 50 C.C. of the heated
culture together with O-2 C.C. of blood broth culture of the living R strain
of Type II. Six mice were similarly injected with the heated culture alone,
four receiving the deposit of 50 C.C. and two the deposit of 100 C.C. of the
broth culture.

All of the above mice were killed seven days later; cultures made from
the seat of inoculation were completely negative in the case of the control
mice and all of those receiving the mixtures except four which yielded a few
R colonies only.

Failure of attempt to in&e reversioa in vitro. Two tubes containing heated
suspension (the concentrated deposit of 100 c.c.) in serum were used as
culture media. They were sown with the R strain of Type II to ascertain if
it could be changed into the S form in. vitro. After incubation overnight
plates were made and the tubes were then centrifuged. The supernatant
serum was pipetted off, leaving the deposit to which was added a fresh quantity
of sterile bovine serum. This procedure was repeated eight times and the
final as well as the intermediate plate cultures yielded only R colonies.


-.- -

152         Ptteulttococcal Types
The whole of the two deposits. each containing the heated Type I pneumo-
cocci from 100 C.C. plus the growt,h of the R strain, were inoculated into two
mice. These were killed 13 days later and found to be healthy.
Importance of the presewe of all the products in the smooth culture. There
was one tube of Type I suspension remaining from which the bovine serum
added a week earlier had not been removed. This was inoculated into a
mouse toget,her with O-25 C.C. of an 18-hour old blood broth culture of the
R II strain. The mouse was killed when ill 3 days later and smooth virulent
Type I pneumococci were grown from the blood and from the subcutaneous
seat of inoculation.

The negative result in the first part of the experiment may have been due
to the removal of some important substance from the heated cult,ure deposit.
As ment,ioned above, 1 C.C. of sterile bovine serum was added to each tube of
deposit in order to make it a favourable medium for the growth of any viable
pneumococci possibly remaining. The total bulk, being now over 1.5 C.C. and
being mainly serum, was too great for subcutaneous inoculation into a mouse;
so each tube was centrifuged, the supernatant serum was removed and salt
solution was substituted.

The experiment shown in Table IX was done to test the hypothesis
suggested above.          Table IX.
                                   Type of culture
     Killed S         Living R     No. of               obtained from
   pneumococci       pneumococci    mouse    ResuIt          mouse
Type I heated 13 hours at  R 4, Type II. Dose  817  Killed 13 days   None
60' C. Dose =deposit of   =0.25 C.C. of blood  818      13 ,, 9,
70 cc. in salt solution     broth culture      819  D;:d 3 ,,   ? S colonies, Type I
             iXX& Killed 13 ,,    None
                                        13
Supernatant bovine serum  Aa above         826 Killed 16 diys  Nke
removed from the above              827 ,. 16 ,, ,,
deposits                       8% ,, 16 ,, ,,
                           829 3, 16 ,, 9,
                               830  D::d  16 ,>
Type I heated as above.   As above         821      4 days  S'iolonies, Type I
Dose = deposit of 70 C.C.                 822
with bovine serum                    823 K%ed 1; 1:   Non: "
                          624 3, 13 ,, 9,

A glucose brot,h culture of Type I, after concentration by centrifuging,
was heated to 60" C. in a sealed tube for half an hour and again for one hour.
The whole of the culture (700 C.C. reduced to 5 c.c.) was distributed in small
tubes, O-5 C.C. in a tube, and to each was added O-5 C.C. of sterile bovine
serum. The tubes were incubated overnight at 37" C. and each was plated;
they were plated again after a second incubation. All were sterile. The tubes
were then divided into two sets. Five were centrifuged and the deposit was
separated from the supernat'ant serum broth. Four of the remaining were
retained whole.

To each of the 14 tubes thus obtained O-25 C.C. of the attenuated R strain
was added and the mixtures were injected into mice.


               F. GRIFFITH               153
Two of the four mice which received the whole culture died of pneumococcal
septicaemia and yielded S colonies of Type I.
One of the five mice inoculated with the deposit resuspended in salt
solution died and numerous small colonies, not definitely smooth, grew from
the blood. These colonies, however, formed firm masses with Type I serum
and one colony subculture killed a mouse from which typical S colonies of
Type I were obtained.

There is a strong suggestion that the whole deposit is more effective in
converting the R strain into a virulent form than the deposit from which
the serum was removed after incubation, i.e. the deposit which had been
washed with serum. The supernatant fluid, however, did not contain any
substance in sufficient concentration to increase the virulence of the R
culture. Although the experiment is not decisive, it rather indicates that
the essential material for the building up of a virulent form from the R form
may be associated with the capsule of the pneumococcus and may be to some
extent washed off.

Conditions aflecting the eficacy of the S antigen in inducing reversion. It is
certain that the efficacy of the S antigen in providing the conditions necessary
for recovery of virulence is variable and, for example, differences in tem-
perature and period of heating may exert considerable influence, as will be
seen from the following experiments.

In Table X are given the results of heating the S culture at 60" C. for
different periods, ranging from 15 minutes to 50 minutes.
Each heated deposit alone was injected into 3 mice, while 2 mice received

Table X.

     Killed S         Living R
   pneumococci       pneumocncci
Type I. Dose = deposit of   None
100 C.C. glucose broth ,,
culture heated 15 mins.    ,,
at GO0 c.
As above          R 4, Type 11. Dose
                   =0.25 C.C.

Type I heated 25 minutes   None
at 60o C.               I?
                    1,

No, of
mouse
978
979
980

994

995
981
982
953

  Result
Killed 13 days
1,   13  9,
I,   13  ,,

Died   2 days

,,    3  1.
Killed ;i days
,*
0   13 ::

Type of culture
obtained from
    mouse
None
9,
I,

S colonies, Types II
and I
S colonies, Type I
None

9,
0

As above          R 4, Type II

996   Died  3 days  S colonies, Type I
997 I, 2 I,       PI   Type II

Type I heated 40 minutes   None
at 60" C.               ,,

As above     R';, Type II

Type I heated 50 minutes   None
at 60" C.               II
                    ,,
As above          R 4, Type II

984
985
9SG
998
999
987
988
959

Killed 13 days
9,
Di:d :4 2 ::
    days
I,    2  0
Killed 13 days
,,
,P

1000  Killed 12 days
  1   Died 3
  (Pn. in blodh)

None
,,
r,
S colonies, Type II
   ,f   Type I
Kane
I,
Nb'ne
Culture overgrown


154         P~zemzococcal T?ypes
the combined inoculation. All the control mice were killed 13 days later and
plate cultures from the subcutaneous seat of inoculation remained sterile.
Culture tests also showed the heated culture to be st.erile.
On the other hand, all the mice, except one, inoculated with the attenuated
strain in addition died of pneumococcal septicaemia within 3 da.ys. The
mouse which survived had received the deposit heated for 50 minutes. The
fellow mouse died and the culture from the blood beca,me overgrown. The
infection was most probably due t,o Type II pneumococci, since microscopical
examination of the blood showed diplococci with well-marked capsules. (AS
a rule a Type I pneumococcus appears in the mouse's blood in short chains
and t,he capsules are rarely as large and well stained as those of Type II
pneumococci.)
The S culture heated for 15, 25 and 40 minutes had almost identical effect
on each pair of mice, one yielding a virulent Type I culture and the other a
virulent Type II. From one mouse injected wit'h the 15 minutes heated
deposit Type II colonies were grown from t,he blood, while on the plate
from the local lesion among the majority of translucent Type II colonies a
single slightly opalescent colony was identified as Type I.
The above results are more striking t'han the examples already given with
cultures heated at 60' C. for an hour or more and there is an indication of a
falling off as the period of heating approaches the hour.
An experiment was therefore made to compare at the same time cultures
heated for over and under an hour.
A virulent Type I culture was grown for a few hours in 2 litres of glucose
broth. It was then centrifuged and the deposit was collected into tn-o sealed
tubes each cont.aining 10 cc. Both were heated in a water-bath at 60" C. for
half an hour. The next day one was heated to the same temperature as before
for a further period of 2+ hours. The tubes were then opened and the thick
suspension was distributed into tubes in 1 C.C. quantit'ies to each of which
was added 0.25 C.C. of blood broth. The tubes were incubated overnight and
plated the next day. One had become contaminated with a bacillus; the rest
were sterile. The heated cultures alone were not tested on mice. Ten mice
were inoculated with the culture heated for 3 hours and nine with the portion
heated for half an hour. Each mouse received the deposit of 100 C.C. together
with 0.25 C.C. of the attenuated R strain of Type II.
The results showed very definitely that there was a considerable difference
in chara,cter between the two differently heated suspensions.
Of the ten mice which received the 3-hour heated culture three died of
septicaemia and S colonies of Type II were grown from the blood. One died
prematurely (3 days) and a few R colonies were grown both from the blood
and from the local lesion. The rest were killed in 6-7 days and R colonies
only were obtained from the seat of inoculation.
Of t,he nine mice which received the +-hour heated culture five died of
septicaemia in 3-6 days and S colonies of Type I were grown from t,he blood.


F. GRIFFITH

One mouse died in 4 days ; the blood yielded S colonies of Type II, but
among these was observed a colony a little whiter than the rest. A subculture
of the latter inoculated into a mouse intraperitoneally produced septicaemia;
a culture and the peritoneal washing gave positive agglutino-precipitation
with Type I serum. The seventh mouse died in 4 days of Type II septicaemia.
The remaining two were killed in 6 days; they showed nothing micro-
scopically in the blood and a few R colonies only were grown from the seat
of inoculation.

The question of intermediate stages in the transformation of type. An attempt
has been made, from time to time, to ascertain whether, if it is the case
that the virulent S form is built up from the R form, the change is gradual,
i.e. whether the R form before developing the virulent S form of another
type passes through a stage when, though still R, it resembles the R of the new
type. The only way to differentiate with certainty between the R strain
derived from Type I and the R strain derived from Type II is by producing
reversion to the S form of the original type without the assistance of any
heated S antigen. The experiment just described offered a suitable occasion
for testing this point. An R colony from the local lesion from one of the
above mice which died of Type I septicaemia was grown in 100 C.C. of glucose
broth. The centrifuged deposit was divided equally between two mice by
subcutaneous inoculation. One died of Type II septicaemia, the other re-
mained unaffected. The R colony chosen was therefore the same as that
inoculated, viz. the R colony of Type II. Colonies were also taken from five
of the mice in which no virulent S form had made its appearance and they were
treated as above. Two of the colony cultures, inoculated in 50 C.C. doses, caused
Type II septicaemia in one of each pair of mice; all the other mice survived.

Positive evidence of an intermediate R form between the attenuated
Type II and the possibly newly formed virulent Type I thus fails.

Further experiments on the destructive effect of heat on Type I S antigen.
The effect of heating the S culture to temperatures above 60" C. has been
given some attention. It has already been noted that Type I virulent culture
after exposure to 100" C. loses the property .possessed by cultures heated to
60" C. of restoring the virulence of the R strain of Type I inoculated at the
same time and does not cause the appearance of S cultures of Type I when
injected along with an attenuated R strain of Type II.

The results of injecting Type I culture heated for 15 minutes at tem-
peratures ranging from 60" C. to 80" C. are given in Table XI. Only the
deposit heated at 60' C. was tested on mice for the presence of viable pneumo-
cocci. Four mice received each the deposit of 65 C.C. of glucose broth and
were killed 10 days later.  Plates made from the seat of inoculation were
sterile; the subcutaneous tissue from the groin of each mouse was removed,
emulsified and injected into a second mouse intraperitoneally ; all these mice
survived. It seems clear that no viable pneumococci remained in the culture
heated to 60" C. for 15 minutes.


156

     Killed S
   pncumococci        Living R    No. of           Type of culture
                                             obtained from
                pneumococci    mice     Result         the mouse
Type I heated at GO'C.   Xone            4               None
for 1.5 minutes. Dose=              Killed 10 days
deposit of 65 C.C. of cul-
ture

Type I as ahove. Dose =
deposit of 30 C.C.

T~III: I hcatcd at, 65" C.
for 15 minute. Dose -.
deposit of 6.5 c.v.
Type I heated at 7O'C.
as above

Type I heated at 75'C.  R 4, Type II, as
as above              above

Type I heated at SO" C.
as above

R 4, Type II, as
above

R 4, Type II. Dose
=@25 C.C. of blood
broth culture

R 4, Type II, as
nl~ovc

2  Died 3 and 5 days  S colonies of Type I
               from blood

4  Killed 10 days    None

4  Killed 10 days (2)  None (2)
  Died 4.5 days (2)  S colonies of Type
            II (i)
           S colonies of Types
            I and II (1)
4  Killed 10 days    ?iOWZ

4  Killed 10 days (3)  None (3)
  Died 3 ,,  (1)  R colonies only (1)

Roth mice inoculated with the R strain of Type II together with the
deposit of 30 C.C. of this broth culture of Type I heated at 60" C. developed
Type I sept'icaemia.

The results with the R Type II plus Type I culture heated at 70" C.
were interesting. Two mice died of pneumococcal septicaemia. S colonies
of Type II were grown from the blood of one and S colonies of Type I from
the other. The local lesion of the latter mouse was plated and among a
majority of R colonies a few S colonies grew, two of which were tested and
found to be Type II.

From the remaining mice, most of them being killed healthy, no S cultures
were obtained.

The Type I cultures heated at different temperatures were a,lso tested on
mice together with an R strain derived from Type I; the dose of heated
culture was only half the amount used in combination with the R strain of
Type II. All the mice which received the culture heated at 65" and 70" C.
respect,ively died of Type I septicaemia. At 75" C. the culture wa.s effective
in causing reversion to the S form of Type I in two out of three mice, whilst
the culture heated at 80" C. produced no effect, the mice remaining well
and the R pneumococci having disappeared from the seat of inoculation.
The destructive effect of the increased temperature on the Type I antigen is
thus exhibited as in the preceding experiments.

These experiments may be summarised as follows.
The inject'ion of heated virulent S culture of Type I into the subcutaneous
tissues of mice together with an attenuated R skain derived from Type II
apparently results in the conversion of the latter into a virulent S culture of
Type I or of Type II. Cultures heated for 15 minutes at 60" C. are more


                F. GRIFFITH               157
effective in producing the transformnt,ion than cultures heated for Iongc*r
periods at 60' C. or at higher tempcraturcs.
The chances of an R strain of Type II reverting to its original S form or
being converted into the S form of Type I after inoculation together wit,h
virulent Type I culture heated at 60" C. for 15 minutes appear to be about
equal.

At 80" C. the active substance in the Type I pneumococcus is so much
altered that the heated culture causes neither reversion of the R form of
Type I to its S form nor transformation of the R form of Type II into the
S form of Type I.

Cultures of Type I heated at 70" C. and 75" C. respectively are still effective
in producing reversion of the R form of Type I, but transformation of the
R form of Type II is rarely brought about by cultures heated higher than
60" C. One positive result with culture heated at 70" C. for 15 minutes was
obtained.

Neither reversion to the S form nor transformation of type has been
obtained in vitro.

Inoculation experiments with heated virulent Type III culture and (1) an
attenuated R strain derived from Type I, and (2) an attenuated R strak of
Type II.
The Type III culture used in the experiments shouTn in Table XII was
grown for a few hours in a flask of glucose broth which on removal from the
incubator was immersed in a water-bath at 60' C. for one hour to arrest

Table XII.

                                    Type of culture
     liillcd S         Living R     No. of              obtained from
   pneumococci      pwumococci    mouse    Result        the mouse
Type III heat.ed 2 hoursat  R 4, Type II. Dose   869   Killed 14 days   None
60' C. Dose =deposit of   =0.25 C.C. of bIood   870      14 I.
100 C.C. of glucose broth   broth culture      871  D;:d   7  ,,  S zolonies,Type III
culture                             872       10  ,,       9, ,,
                                 873  Kliled 14  ,:              "
                                 874  Dt:d  14  ,.   Non:'
                                 875        2  ,,   S colony, Type III
                                876    9,    8  7,   S colonies, Type III
Type III, as above     R 3, Type I. Dose   877  Killed l-1 days   None
                 =O.% C.C. of blood   878
                  broth culture      879   :: :a ::  S golonies, Type III
                                880    1,   14  ,t    None
                                  881    ,,   14  ,,     9,
                                 882    ,,   14  ,,     ,,
                                 883    ,,   14  ,>     ,,
                                 884  Di':d  14  ,,
Typo III steamed at  R 4, Type II. Dose   885      3 days S I?olonies, Type II
100" C. for 12 minutes.   =0.25 C.C.        886   Killed 14  ,,    None
Dose =deposit of 100 C.C.                 887  D;;d  14  I,
                                 888        3  ,,  S yolonies, Type II
Type III, as above     R 3, Type I. Dose  889  Killed 14 days  R colonies only
                  =0.25 C.C.        890   Died 1"      None
                                891   Killed 14 ::
                                892    ,,   14  ,,   R"colonies and nnc
                                   S colony


L

158         PueunaococcaE Types
autolysis. On the following day the 2400 C.C. of broth were concentrated by
cent,rifuging to 24 cc. ; 16 cc. were placed in a sealed tube and heated for
one hour at 60" C., and the remaining 8 C.C. were steamed at 100" C. for a
full 12 minutes.

In this instance mice were not injected with t.he heated culture alone.
(a) Heated to 60" C. Eight mice were inoculated with the R strain of

Type I and eight with the R strain of Type II, both a.ccompanied by a dose
equivalent to 100 C.C. of Type III S cult,ure heated t,o 60" C. It will be seen
from Table XII that the watery colonies of Type III appeared more frequently
in the mice inoculated with t,he attenuated R strain of Type II t,han in those
which received, the R strain of Type I, viz. five times in the former and
once in the latter. This fact lends some support to the view that the par-
ticular type of R strain is the important factor in the production of the S
colonies of Type III. Incidentally it is further evidence against the hypothesis
t'hat viable Type III pneumococci persisted in the culture after heating.

The low virulence of t,hese newly formed Type III pneumococci, as one
might term them, is noteworthy.  The mouse inoculated with the R strain
of Type I and the heated Type III culture was apparently well when killed
14 days later, and the S colonies came to light on t,he plate made from the
subcutaneous seat of inoculation. These S pneumococci, which were in pure
culture, were certainly multiplying in the loca.1 lesion and, though evidently
of low virulence, might ult,imately have caused the death of the mouse. Some
of the Type III pneumococci from the mice inoculated with the R strain of
Type II showed more virulence, since three out of the five positive mice died
with pneumococcal septicn.emia in 7-10 days. Mouse 875, which probably
died prematurely from shock, yielded a single S colony among numerous
R colonies from the seat of inoculation; the blood culture was contaminated.
The fifth mouse, 873, was, like that in the first menbioned series, perfectly
well when killed, and S colonies were grown from the seat of inoculation.

The large watery colonies of Type III on blood agar plates were very
typical in appearance, and, in addition, the blood sinears from the mice which
died showed the characteristic picture of round cocci with large well-stained
capsules. I have had to rely for the identification of Type III pneumococci
on the above appearances in conjunction with negative agglutination reac-
tions to both Types I and II sera, since, in spite of several trials, I have not
recently been able to prepare a serum which gives the characterist,ic agglutina-
tion reaction with a virulent Type III culture.

(a) Heated to 100" C. The steamed Type III culture injected with the
attenuated R stra.in of Type II caused the latter to revert to the virulent S
form in two out of four mice.

An interesting result was obtained in one of the four mice which received
the R strain of Type I together with the steamed Type III culture. The
plate culture from an abscess at the seat of inoculation produced numerous
small apparently rough colonies and one typical smooth disc-shaped colony.


F. GRIFFITH

159

Subcultures from the latter were virulent for mice; pneumococci multiplied
in the capsulated form in the blood, and cultures from the blood yielded
typical smooth discs. I was unable to identify the strain as Type III, since
the typical watery colony was not produced; also no agglutination was ob-
tained with Types I and II sera or with any of the available group IV sera.

In either case, whether it is a Group IV strain or a Type III, the result is
important enough to encourage further experiments with steamed culture.
One must however bear in mind the possibility of accidental contamina-
tion, especially in view of a somewhat similar circumstance which is recorded
in the description of the next experiment.

A virulent Type III culture in glucose broth wm heated for 3 hours at 60' C. in a sealed
tube and was injected subcutaneously into seven mice together with an R strain of Type II.
The dose of the former w&s the deposit of 70 C.C. and of the latter 0.2 C.C. of blood broth.
Four mice died in 4-7 days of pneumococcal septicaemia and watery colonies of Type III
were grown from the blood; the remaining three mice were killed in 7 days and in each
case similar colonies were grown from the seat of inoculation. The above wits an excep-
tional result and fortunately an equal number of mice were injected with the same dose of
the heated Type III culture alone. All the latter seven mice were killed on the 7th day
after injection and cultures were made from the seat of inoculation. All the plates were
sterile with one exception in which a pneumococcus colony appeared.

(In view of this disconcerting result in one of the control mice, I will describe in detail
the procedure in making cultures from the seat of injection.
The mouse is pinned out and the abdominal skin is seared with a hot iron, but not
exactly over the seat of injection. Through the seared area an incision is msde with a
sterile knife and the edge of the skin is grasped with a pair of forceps. The skin, held
firmly, is reflected back until the seat of injection is exposed and is then pinned down.
A fresh pair of sterile instruments is used to scrape the subcutaneous tissues and usually
the gland in the groin is removed as well. The material is placed in a small tube with a
few drops of broth in which it is rubbed up. A loopful of the fluid is spread on a plate
and to the remainder a little blood hroth is added. If the next day after incubation the
fluid culture shows on microscopical examination of smears any diplococci it also is
plated.)

In the case of the mouse referred to above there were no diplococci in the tissue fluid
after incubation and the plate made subsequently from it produced no growth. The plate
direct from the tissue emulsion was sterile except for a single smooth disc shaped colony
which was bile soluble and virulent for mice. At no time did this strain produce watery
colonies like Type III and there was no agglutination with Types I and II sera.
This result occurred some three months before the writing of this article and in spite
of numerous tests designed to reveal the presence of any viable pneumococci in heated
cultures I have never again found any evidence of viability either by culture or through
the mouse, even in cultures heated at 60' C. for so short a period as 15 minutes.
On the whole I am inclined t,o think that the pneumococcus responsible for the colony
fell on the plate while it was being spread.

Still it is often useful to record unexpected occurrences when there is no absolutely
certain explanation. A result which appears to the worker concentrated on a particular
issue to be a regrettable flaw in his working may be significant to another considering the
subject from a different point of view.

These experiments may be summarised as follows:
The injection of S culture of Type III, heated at 60" C. for two periods


160         Pneunzococcal Types
of one hour each, along with living R strains derived from Type I or Type II
results in the appearance of an S pneumococcus of Type III.
This transformation of type occurs more frequently with the R form of
Type II than with the R form of Type I.
The newly formed strains of Type III sometimes kill the mice from
septicaemia in 7-10 days and at other times are only discovered at the seat
of inoculation when the mouse is killed, apparently well, 14 days after inocu-
lation.

Inoculation experiments with heated virulent Type II culture alzd di$erent
        attenuated R colony strains of Type I.
The results of inoculating attenuated R strains of Type I together with
heated Type II culture are shown in Table XIII. A comparison is made
between different R colonies (developed on a plate from a virulent Type I
culture grown in homologous immune serum).
The R strains were also inoculated into mice (1) alone, (2) together with

Table XIII.

     Killed S
   pneumococci
Type II heated 2 hours at
60o c.  Dose =deposit of
90 C.C.

Type III heated 2 hours at
60' C.  Dose =deposit of
80 C.C.

Type II heated 3 hours at
60" C.  Dose = deposit  of
90 C.C.

Type III heated 3 hours at
60" C.  Dose = deposit  of
80 C.C.

Living R
Type 1
pneumococci

R colony 1
  9.    1
  I,
  ,,  ;
  ,f
  I,  I

,, 4
,, 4
,f 5
  ,I ,, :
     6
R c:louy 1
  9, 1
  >> ,, i
  ,,

  I,  3"
  ,, 4
  0 4
  ,, 5
  ,, 5
  I, 6
      6
R cb;ony 1
  ,9 ,, ::
9, i
  ,,
      3
R ciiony 4
  ,, 4
  I, 5
  ,, 5

9, ,, x

No. of
mouse
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759

  Result
Killed ;% days
Diid  11 :I
,,   10  ,,
I,    4  ,9

K&d 11 4 ::
Died   4  ,,
Killed 13  ,,
Di';d : ::
Killed 13  ,,

Killed 11 days
,,   11  ,,
3,   9
9,   11 ::
9,
I, 1: ::
,I   10  ,I
9,   11  1,
,,   10  ,,
,f   11  ,,
,,   11  1,
,,   9
Killed 12 dzys
>I   12  ,I
,,   13  1,
,,   6  ,,
Died   3  ,,
Killed 13  ,,
Killed 10 days
,,
,,  ii ::
I,
,, 1; ::
3,   10  ,I

Type of culture
obtained from
the mouse

None

S Colonies, Type II

  ,,      ,>
  1,      ,,
  ,,      ,,
  ,,      1,
  ,,      ,,
  ,I      ,t
  1,      I.
Non: "
None

I.
,>
,t
,I

S colonies, Type III
None
I,
1,
I,
Nine
I,
S iolonies, Type II
Non: "
None
S colonies, Type III
NOW

77
I,
9,


               F. GRIFFITH               161

heated Type I culture, and (3) with heated Type III culture; the results with
the last only appear in the table.
The R strains were each tested subcutaneously on two mice in a dose of
about 1 C.C. of blood broth culture; all the mice were well when killed in
9-16 days and no pneumococci were recovered from the seat of inoculation.
The virulent cultures were killed by heating for two hours at 60" C. ; in
the case of Types II and III, cultures heated for 3 hours were also used.
As a test of sterility the Types II and III cultures (heated 2 hours) were
injected into two mice. Each mouse received subcutaneously the deposit of
130 cc. of glucose broth; both remained well and were killed 16 days later.
Cultures from the seat of inoculation remained sterile; in one of the two
mice t,he culture was made from an encapsulated abscess which had formed
under the skin and still contained fairly well staining diplococci.
From the majority of the mice inoculated with t,he mixture of the above
heated culture and the living R strains of Type I, virulent S colonies of
Type II were obtained. In the case of the R colony 1 both mice were negative
while only one of the pair inoculated with R colony 6 yielded S colonies of
Type II.
It is interesting that of all the mice inoculated with the twice heated
Type I culture together with the R strains only the one which received
R colony 1 failed to develop fatal Type I septicaemia. This result certainly
suggests that R strains may differ in their suitability for mutation in the
same Kay as they differ for reversion experiments.
There is a similar indication in the experiments with Type III heated
culture. Only one R strain, R colony 4, yielded the watery colonies of Type III
and the posit,ive results occurred with the suspension heated for two hours and
three hours respectively.
It will be noticed that the heating for an additional hour has lowered the
proportion of positive results with the killed virulent Type II culture. The
experiments with heated Type II culture and attenznted R strains derived
from Type I have not always been so successful as the above in producing
an apparent, change of type, as will be seen from the following example.
A suspension of virulent Type II was heated for one hour at 60" C. and
again for a second hour and was injected into mice (doses = deposit of 90 c.c.)
together with (1) R colony 3 culture of Type I, (2) R colony 2 culture of
Type I.
Eight mice were used for each R strain and one mouse out of each series
died of Type II septicaemia in 4 and 10 days respectively. Excepting one
which died prematurely and one which died in 8 days (only R colonies at
the seat of inoculation) the rest were killed 16 days after inoculat,ion and no
pneumococci of any form were obtained from the subcutaneous tissues.
In another experiment the Type II S culture was heated for three hours
at 60' C. and was injected in doses equivalent to 60 C.C. of broth culture.
Four mice were injected with the heated cultures alone and were killed in


--

16.2         Pm2mzococcal Types
10 to 14 days; plate cult'ures from the sea.t of inoculalion yielded no pneu-
mococci.

The attenuated R cultures of Type I employed in this experiment were
different from those used previously. They were six different colony strains
from a plate from the fourth successive cuhure of Type 1 in homologous
immune serum. Three mice were inoc,ulated with 0.25 C.C. of each (t,otal of
18 mice) together with t'he above mentioned heated culture. Two inoculated
wit'h different R colonies were killed, ill in 7 and 10 days respectively, and
S colonies of Type II were grown from t,he blood. The remaining 16 were
killed when well after 10 to 14 days and cultures from t,he subcutaneous seat
of inoculstion were negative except in three instances where R colonies alone
were grown.

These experiments may be summarised as follows:
The injection of virulent S culture of Type II killed by heat a,t 60" C.
together with living R st,rains of Type I has resulted in the formation of a
virulent S culture of Type II.

The transformat'ion has t,aken place when t,he virulent culture has been
heated at 60" C. for 2 and 3 hours respectively, but. the positive results were  1
less frequent in the case of the culture heated for bhe longer period. Diffcrcnt
R strains appear to vary in their ability to develop into a new S form under       '

t'he influence of the heated virulent culture.                 i

Inoculation experiments with heated virulent culture of Types I and II together
       with living attenuated strains of Group IV.
The living R strain of Group IV in the experiments in Table XIV was
derived from Type II A by growth in homologous immune serum. The latter

   Killed S
pneumococci
Type I hrat,ed at
tiO" C. for 2 hours

Type II heated as
above
Type II d heated
as above
NOlIt2

Type I heated at
GO0 c. for 2 hours

Type II heated at
tio" c. for 2 hours

Table XIV.

Living R

pneumococci
R 1, Type II .4. Dose
=0.25 cc. of blood
broth culture
R 1, Type II A, as
above

R 1, Type II A, as
above

R 1, TypeIIa. Doses
=0.5-1.0 C.C. of
blood broth culture
R 1, Pn. 41. Dose
=0.2 cc. of blood
broth culture
R 1, Pn. 41, as above

No. of
mice
5

5

5

Result

All died in 2-5 days

All died in 2 days

All died in 3-5 days

All survived

1 killed in 9 days   S cultures of Pn. 41
4 died in 4-8 days    from 4, nil from 1

2 killed in 9 days   S cultures of Pn. 41
3 died in 3-G days   from 4; nil from 1

Type of culture
obtained from
   mice

S culture of Type I
from each

S culture of Type II
from ea.ch
s culture of Type IIA
from each _

-

Pn. 41  heated  as    R 1, Pn. 41 as above    4   3 died 2-4 days     S cultures of Pn. 41
above                      1 died prematurely
None            R 1, Pn. 41.  Dose   3    1 killed in 9 days   S culture of Pn. 41
               =0.6 C.C. to 1 C.C. of     2 died in G-7 days   from 2; nil from 1
              culture


F. GRIFFITH

163

was effective in producing attenuation since none of five colonies selected
reverted when inoculated subcutaneously in mice in doses of 10 c.c.; R colony 1
\vas chosen for this experiment and three control mice were inoculated.

The results shcw that the R st,rain of II A was readily transformed either
into t,he S form of Type I or into t'he S form of Type II, and that reversion
t,o its original S form occurred when it was inoculated with heated culture
of t,hat S form. The R strain of II A inoculated alone has not reverted,
though larger doses than 10 C.C. have not been tried.

The second rough Group IV strain, Pn. 41, gives an interesting result
and, in addition, provides a useful control for the heated cultures since, as
will be seen, t'he heated Types I and II suspensions which were the same as
those used \vith the rough Type II A never caused the appearance of an
S strain either of Type I or Type II, thus showing that the heating had been
effective in killing t,he S cultures.

There was no transformation of t,he R strain of Pn. 41 and this fact may
perhaps be connected in some way with the insufficient attenuation of the
strain which, as will be observed, reverts readily unaided. The R colony
culture used was from one of four colonies which were picked of? the plate
sown from the culture in homologous serum. Evidently the serum was weak
in protective substances, since two out of the four reverted on the pre-
liminary testing. R colony 1 which failed to revert when first tested, also
reverted too readily unaided when tested later.

    Inoculat,ion of living and dead R cultures.
The esperiments in Table XV are negative with one exception where an
R strain derived from Type II reverted to the S form of II when inoculated
into a mouse together with heated rough Type I culture.
The experiments were repeated except that six mice were used in each
series = totJal of 24 mice. All of the mice survived.

Table XV.

                                  Type of culture
    Killed El        Livin,o R     No. of                obtained from
  pneumococci      pneuniococci    mice      Result         the mouse
Rough Type I heated                  3   Killed 16 days (2)   NOIN2
at GO" C. for 2 honrs.  R 5, Type II. Dose
               =O.P.j C.C. of blood     Died 4 days (1)   S colonies of Type II
Dose = deposit of 100   broth culture
C.C. of broth culture
As above        R 3, Type I. Dow   4   Killed 16 days     Kone
                 =0,5 e.c.
Rough Type II, as  R 4, Type II. Dose 4    ,, 16 ,,        9,
above             =0.23 C.C.
As above        R 3, Type I. Dose 3    ,, IF ,,        ,,
                =0.25 C.C.

The heated R culture, although the doses were very large, viz. the
deposit of 170 C.C. of brot,h, exerted no effect on the living R strains either in
the direction of reversion or transformation of t,ype. This is consistent with


lG4         Pnemmoccccl Typs
t,he view that in bot,h cases t'he result depends on the presence of S antigen
of which there are only traces in an R strain. The heating to which the
R culture had been subjected would diminish the activity of t,he small amount
still further or destroy it ent.irely.

III. DISCUSSION.

The serological ana,lysis  o f a bacteria~l species has an obvious practical
a,pplication in bact.eriological diagnosis a,s well as in the preparation of anti-
bacterial therapeutic sera. There are! however, other issues, probably of
greater importance, which have to do with the occurrence and remission of
epidemics, the a,ppearance of epidemic t>ypes in certain diseases and the
attenuation of the infecting agent in others. It must ha.ve occurred to every
serologist to ask himself the meaning of the types he has defined. Do sero-
logical types represent stages in the norma! life history of a bact,erium or are
t,hey the response on the part of t,he bact'erium to changes in the immuno-
logical state of the animal host? If it is a question of altered environment,
are the influences which initiated the divergence of type still at work, i.e.
are the type characters still in a state of flux, or have the different varieties
become stabilised?

On considering the above questions one cannot fail to realise that their
solution would be a valuable contribution to the epidemiology of disease and
would explain some of the phenomena in the rise and fall of epidemics. In
certain bacterial infections, of which lobar pneumonia serves as an example,
it is possible t'hat even the most potent antisera will not avail to cut short
t,he disea,se once the organisms are established in the tissues. Attention must,
therefore, be direct.ed to prevention of infection: and to this end a close study
of bacteria,1 virulence and its relation to variations in serological type is
essential.

Virulence and type characters are closely related in the pneumococcus.
When pneumococci are grown in homologous immune serum, some descendants
become attenuated in virulence and these can be recognised by their formation
on solid media of a distinctive va.riety of colony known as the R form of the
pneumococcus. The virulent or S form of pneumococcus produces in fluid
media, and still more abundantly in the peritoneal cavity of the mouse, a
soluble sub&ance which, though not itself antigenic, gives a copious preci-
pitate with the appropriate antiserum. Each type of pneumococcus forms a
special soluble substance which has no affinity for an antiserum prepared
with any ot,her pneumococcal type and it is to this property that the re-
markably clea)r definition of the serological races of pneumococci is due.
These substances have been shown1 to consist chiefly of carbohydrate and,
though highly reactive, t.o be non-antigenic. As a result of its change to the
R form, the pneumococcus genera.lly loses this power of producing soluble

1 H4elberger, AI. and Avery, 0. T. J. L'XJL JfccZ. 38, 73 anti 40, 301.


                F. GRIFE'ITH                105
substance, though individual strains differ in the degree of this loss. In addi-
tion to the exceptional R variety of a Group IV strain described pp. 116, 117,
which produced a considerable amount, and was virulent for mice, I have shown
that quite attenuated R strains ma>- form traces in the peritoneal cavity of
the mouse. Cultures of R strains, however, rarely contain sufficient soluble
substance to give a demonstrable precipitation with the appropriate ant,i-
serum, and, in consequence, the agglutination test no longer serves to identify
a strain with the virulent type from which it was derived. Since virulence
and the capacity to form soluble substances are at,tributes of t.he S &rain,
their possession may for convenience be ascribed to a special antigen which
may be termed the S antigen.  Thus an attenuated R strain which has no
demonstrable S antigen has lost, the serological characters of its type, but,
if virulence is restored by passage through mice, the strain reverts to the
S form of the type from which it was derived.
Some att.enuated R strains revert readily to the virulent form and this
feature is correlated n-ith demonstrable traces of S antigen in t.heir com-
position. Ot.her strains have been found in which the R st,ate is much more
stable. In a series of peritoneal passage experiments beginning with one
strain and carried on with its descendants reversion has occurred in one
branch of the descent and not in another.

The ncquiremcnt of the typical characters of a virulent pneumococcus by an R strain
from which the S antigen has been almost eliminated by growth in immune serum recalls
some experiments by Bail on the anthras bacillus'. By exposing a culture of anthrax
bacilli to a ternpcmture of 12' C. he obtained strains which Tvere almost deprived of their
power of producing capsules. Such a strain might produce a mixture of cclonies some of
which on subculture invariablv failed to form capsules while others showed a sma,ll minority
of capsule-forming bacilli. This result he ascribed to a deficient inheritance of tfle capsule-
forming substance, so that an individual bacillus was able to endow only one of its
descendants with a sufficient amount to produce R t.ypical capsule-forming strain.
That there might be some principle uncleriSing these infrequent and
apparently haphazard positive results was suggested by the following observa-
tion. Au attenuated R strain which regularly became virulent when inocu-
lated iotraperitoneally into a mouse failed to revert whelt the same dose WiLs
introduced into ;L vein. Apparent,!!- attenuated pneumococci require a pro-
tect,ed situation in which to multiply and acquire virulence, and this they
find occasionally when inoculated into the peritoneal cavity. If they are put
directly into the blood stream in a dose which does not overwhelm the animal,
it \vould appear that they do not find such suitable conditions and are readily
disposed of.
This view has been confirmed by subsequent experiments a,nd it has been
found that a more certain met.hocl of ascertaining whether an R strain is
capable of reverting is by the inoculation of a large dose of cuhure under the
skin of a mouse. The mass of culture, I suppose, forms a nidus in which the

           L Ce~cfraZlJ. f. Lidif. Orig. 79, p. 1-13, 1917.


attenuated pneumococci are prot.ected from the bactericidal action of the
tissues. Since, however, attenuated pneumococci may remain alive and un-
altered in the subcutaneous tissues for two or three weeks, local protection
is clearly not the only factor in this met,hod of restoring virulence. It seemed-l
possible that the mass of R pneumococci, disintegrating under the action of
the animal tissues, might furnish some substance which was utilisable by the
survivors to build up their virulent st,ructure. Acting on the assumption
that this material might be the S antigenii-substance, which in varying
amounts persists in the R form, I inoculated into the mouse's subcutaneous
tissues a very much smaller dose of living R pneumococci together with a
mass of killed virulent culture. The result of this greater concentration of
S antigen, or perhaps of some substance derived from it, was to make the
conditions &ill more favoura,ble and reversion of the R strain to the virulent
form was secured with great regularity.
The observation that a sublethal dose of a bacterium may cause a fatal
effect when inoculated together with the sterile products of that bacterium
has long been known a.nd forms the basis of the theory of aggressins, It
was at first maintained that aggressins could only be obtained from the
bacterium through contact with living animal tissues, e.g. from the peritoneal
exudate of an animal inoculated with virulent culture, though it was finally
conceded that they might be present to a slight degree in disintegrating
cultures.
The action of the killed pneumococcus culture in enhancing the virulence
of the R strain irk viz.0 though not in citro is certainly a.nalogous to that of
the hypothetical aggressin, and these results may throw fresh light on an
obscure subject.
The principle of the action of aggressins was held to be their toxic influence
on the leucocytes which were thus rendered incapable of attack. This is no
doubt partly the function of the mass of killed virulent culture injected to-
gether with the attenuated R variety of pneumococcus, but there are other
considerations which support the view already put forward that the attenuated
organisms actualiy make use of the products of the dead culture for the
synthesis of their S antigen.
An R strain is most readily transformed into t,he S variety when the
killed culture used is of the same serological type as that from which the
R strain was derived. For example, Type II S culture killed by steaming at
100" C. readily causes the R strain of Type II to revert in the mouse, whereas
Type I S culture, similarly treated, does not, though it may when heated to
GO" C. (vide ,i71JTa).
An exception to the statement above is that certain Group IV strains are
practically as effective as Type II in causing the R form of the latter type to
revert to the S variety. The same Group IV strains, however, have no effect
when injected together with an R strain of Type I. Apparently the antigens
of these Group IV strains are more closely related to Type II than to Type I


               F. GRIFFITH               167

and the results are further evidence of t,he specific selective action of different
pneumococcal antigens in causing reversion.
The specific effect of the killed culture is at first sight less evident where
the culture is heated t,o a lower temperature than 100" C. For instance,
cultures of Type I heated at GO', 70" or 75" C. frequently cause the R form
derived from Type II to revert to the S form of Type II. How does this
result affect the hypothesis suggested above that an attenuated R strain
with deficient S substance requires the products of an S culture of the same
type with which to rebuild its former type characters and virulence? One
must consider first the effect of heat on the two types, I and II. Type II
virulent culture, heated for so short a period as 15 minutes at 60" C., has so
far never caused the R form derived from Type I to revert to the S form of
Type I, although steamed cultures of Type II are effective in inducing re-
version of its own R form to the corresponding S form. On the other hand,
Type I while effective after heating at t,emperatures of 60"-75" C. in producing
the R to S change with its own type loses this property when heated at
80' C. or higher,
These observations suggest that the specific S substance of Type I suffers
more by exposure to heat, that is to say, a greater proportion of it is destroyed,
than that of Type II.
BJL_S__su-bstance I mean that specific protein structure of the virulent
pneumococcus which enables it. to manufacture a specific soluble carbohydrate.
This. protein seems to be necessary as material which enables the R form to
build up the specific protein structure of the S form. But it appears that
this material may be modified by heat in such a way that the R form cannot
utilise it for the reconstruction of its own internal structure. (The specific
carbohydrate which is the product of the S form is unaffected by heat.)
In order to reconciie the experimental data referred to above with the
hypothesis that the R pneumococcus which reverts in the mouse to the
S form has synthesised its S antigen from similar material in the heated virulent
culture injected at the same time, it is necessary to assume that a virulent
Type I pneumococcus contains some S antigen of Type II. An alternative
hypothesis would be that the R form of Type II is able to reconstruct its
virulent S form from either the S substance of Type I or that of Type II.
One is then faced with the difficulty of accounting for the failure of an R
form of Type I to build up its S form from Type II S substance.
The amount of S antigen of Type II in Type I must obviously be small
in proportion to the Type I, since the serological tests give no indication of
its presence, and it is legitimate to suppose that heating to a temperature
which would not greatly diminish the tot,al amount of Type II S antigen in
a Type II pneumococcus might conceivably destroy it entirely in a Type I.
This is supported .by the experiments which show that after heating to 80" C.
the capacity to induce reversion of the R form of Type II is lost by the
Type I culture and retained by the Type II culture.


168         Pneunzococcal Types

The relationship between Types I and II pneumococci just suggested,
in which the major antigen of one type is represented as a subsidiary antigen
in the other is not without parallel in other bacterial groups, e.g. meningo-
cocci. There are special circumstances, related no doubt to the formation of
soluble substance, which in the ordinary serological test keep this relat,ionship
in the background.

If there is a reciprocal relationship between Types I a.nd II, a,s one would
expect, what is the expla.nation of the failure of the reversion experiments
to show evidence of the presence of Type I antigen in a Type II pneumo-
coccus? It is. I think, a questsion of the difference in heat resistance between
the two antigens. As Type I antigen is the more heat sensitive, the smsll
amount assumed to be present as a subsidiary antigen in a Type II pneumo-
coccus might be destroyed by a temperature which would not affect the
Type II antigen in a Type I pneumococcus.

These considerat,ions, I think, afford a reasonable explanation of the
experimental data in connection with the restoration of virulence to an
attenuated pneumococcus. The chief points are :-( 1) in the change from the
S to the R form some of the S antigen may persist; the amount, rarely
demonstrable by in vitro tests, varies in different R strains; (3) the S antigen
remaining in an R st.rain may be regenerated and reach its original abundance
under suitable conditions, e.g. inoculation subcutaneously into a mouse in
large doses or in small doses plus a mass of heated culture containing the
particular S antigen; (3) an S strain of one type (I or II) may cont,ain in
addit,ion to it,s major antigen a remnant of the other type antigen.

Application of the principles underlying these observations to the question
of transformation of one type into another has given results of considerable
interest.

When pneumococci of Types I and II sre reduced to their respective R
forms by growth in homologous immune sera, they lose nearly all their major
S antigen though they may retain their minor S antigens whi$h are pre-
sumably not affected by the heterologous immune substances. But the major
S antigen apparemly still preponderates, since an R strain on reversion to
the S form regains its original type characbers. Some R stra.ins, however, do
not revert even when inoculated in large amounts under the skin of a mouse,
and it is not unlikely t,hnt in such strains the major antigen has been reduced
to the same insignificant amount as the minor antigen.

In such circumstances an R strain derived from Type I would be identical
with an R strain from Type II; and under suitable conditions the development
from it of a virulent form of eit'her type might be anticipated.
This has been shown actually to occur; a virulent Type I pneumococcus
can be derived through the intermediary R form from a virulent Type II
pneumococcus, and z-ice versa.

Up to t,he present I have maintained a distinction between the R forms
derived from Types I and II respectively, though, as I have stated earlier,


               F. GRIFFITH              169
they can be identified only if they revert unaided to the S type from which
they originated.
JVhen the R form of either type is furnished under suitabIe experimental
_conditions with a mass of the S form of the other type, it appears to utilise
that antigenas-a pabulum from which to build up a similar antigen and thus
to develop into an S strain of that type. Therefore the R form of Tvpe II,
when inoculated together with a heated suspension of Type I, uses the antigen
of the latter strain and an S pneumococcus of Type I makes its appearance.
There is a further complication since, as it a,ppears, the heated Type I SUS-
pension contains a subsidiary 8 arnigen of Type II, and some of the R
pneumococci may use this to develop an S strain of Type II'. As a result
one mouse may yield the S form of Type I and another the S form of Type II,
while quite frequent'ly the same mouse may yield both types.
Similarly, if the R form of Type I is inoculated together with the heated
S culture of Type II? a virulent S form of Type II is developed. (The S culture
of Type II never causes the R form of Type I to change into the corre-
sponding S form, because, as I have already explained, the subsidiary Type I
antigen is destroyed by the heating.)
These observations suggest that there is no essential distinction between
the two R varieties. In fact, there are certain indications that the R pneumo-
coccus in its ultimate form is the same, no matter from what type it is derived;
it possesses both Types I and II antigens in a rudimentary form or, as it
may be differently expressed, it is able to develop either S form according
to the material available.
If Type III substance is offered as a pabulum, either form is able to build
up a typical S strain of Type III, though it appears that the R form derived
from Type II is more readily converted into Type III than is the R form of
Type I. Why there should be this difference is not clear, t,hough it may be
assumed to have some association with the fact that this particular R strain
from Type II has not reached the same stage of attenuation as the R strain
of Type I. (The latter in the relatively few tests made has not reverted, as
does the R Type II, to the S form when inoculated unaided, i.e. alone in
large doses.) There is also a further point of distinction in that injection of
heated Group IV cultures has caused the R form of Type II to revert but
not the R form of Type I. The S substance of the Group IV strains is
evidently closely related to that of Type II since it provides a suitable pabulum
for the regeneration of the virulent S strain. In this connection I may recal1
that Group IV strains appear in the sputum during convalescence from
Type I pneumonia, and it is suggested that they are formed from the Type I
after suppression of the major antigen through the action of the immune sub-
stances and by the development of the subsidiary Type II antigen, though
not to its full complexity.
It may be that the minor antigen in a Type I is not actually a fully de-
veloped Type II S antigen in small amount but a less differentiated substance


170         P;?teernzococcctl Types
which serves indifferently as a foundation for t.he building up of either Type II
or a Group IV strain.
The R form derived from a Group IV strain, viz. Type II -4, can be trans-
formed into the S forms of Type I or Type II or changed back to its original
S form according to the particular S substance which is injected along with it.
On the other hand, another Group IV strain which was incompletely attenuated
invariably reverted to its original S form no matter what type of S culture
was injected with it. It seems that, if a pneumococcus has a moderately well-
developed S structure, there is no tendency to develop into an S variety of
any other type.
The method by which transformation of type has been secured consists
in heating t.o 60" C. for 15 minutes up to 3 hours a virulent culture of one
type and inoculating a large amount of the heated culture under the skin of
a mouse together with a small dose of the R strain derived from another
type.
Experiments with culture heated at temperatures higher than 60" C. have
rarely been successful in causing transformation of type. In one instance
the S form of Type I was obtained from a mouse which had been inoculated
with t,he R form derived from Type II together with a suspension of Type I
heated to 70" C. for 15 minutes, in a second the S form of Type II was
obtained from a mouse inoculated with the R form of Type I together with
a virulent Type II culture heated to 65" C. for 15 minutes.
The question arises whether heating at the above temperatures had in
fact killed all the individual pneumococci in the mass of virulent culture or
whether t,he apparent change of type was due to the occurrence of a survivor.
I have given this question careful consideration and I have never been able
by the ordinary methods of culture and animal inoculation to demonstrate
the presence of viable organisms in the heated cultures. Since there is no
reason to suppose, and I have had no evidence t,o show, that the R strains
used were mixed, there seems to be no alternative to the hypothesis of trans-
formation of type.
A few years ago the statement that a Type I strain could be changed into
a Type II or a Type III would have been received with greater scepticism
than at t,he present day. Since, however, it has been shown that a pneumo-
coccus can readily be deprived of its type characters and virulence, and that
under favourable conditions these can be restored, the possibility appears less
unlikely.
The apparent transformation is not an abrupt change of one type into
another, but a process of evolution through an intermediate stage, the R
form, from which the type characters have been obliterated. Mutation of
type among disease-producing bacteria is a subject of obvious importance in
the study of epidemiological problems. If it can be proved to occur in the
pneumococcus group with its sharply defined immunological races, the possi-
bility can hardly be denied to other bact,erial groups where the serological


F. GRIFFITH                171

types cannot be differentiated without the help of agglutinin-absorption
experiments.

The position in regard to the causal relation of different types of pneumo-
cocci to lobar pneumonia presents certain difficulties. Types I and II pneumo-
cocci, which cause 60 to 70 per cent. of the total cases of lobar pneumonia,
are rarely found in the normal nasopharynx except in close contacts of the
disease. Whilst this latter observation indicates some power of epidemic
spread, it is not often demonstrable that a case of pneumonia acts as a focus
for fresh cases. On the other hand, Group IV pneumococci are of common
occurrence in the nasopharynx and about 25 to 30 per cent. of pneumonia
cases are attributable to various types in this group; these cases are generally
considered to be of autogenous origin.

It is a very remarkable fact that the incidence of the chief types of
pneumococci in lobar pneumonia is almost identical in countries where the
climatic and social conditions are similar. While this occurrence is not easily
explained on the supposition that the disease is partly infectious and partly
autogenous, it is not inconsistent with the evolution in the individual of
special types most suited to set up pneumonia, i.e. t,he similar distribution
is due to similar composition of the population as regards susceptibility to
the pneumococcus and not to similarity in the diffusion of pneumococcal
types.

In convalescence from pneumonia Types I and II tend to disappear from
the respiratory tract and are replaced by the common Group IV strains.
According to the more generally accepted view, the chief types die out and
the Group IV pneumococci, the normal inhabitants of the nasopharynx, again
come into prominence. An alternative bypothesis which was purely specu-
lative in the absence of evidence of the instability of pneumococcal types is
that the chief types revert to the Group IV varieties from which they were
derived during the development of the disease in the individual.

On the lines of my previous argument as to the process at work in the
development of a virulent S strain from an attenuated R pneumococcus, it
may be surmised that the immune substances formed during recovery suppress
the S antigens of the chief type and under suitable conditions the subsidiary
antigens are developed to form a new virulent type-in this case one of the
varieties of Group IV. As mentioned earlier, I have shown that the sputum
of a case of pneumonia due to Type I almost invariably contains, in addition,
one or more virulent strains of Group IV, and as many as four distinct types
have been isolated from a single case.

This latter instance certainly suggests that the Group IV strains are
variants of the Type I, and it is of some significance in this connection that
the antigens of the Group IV strains have been shown to be related to that
of Type II which is represented as a subsidiary antigen in a Type I pneumo-
COCCUS. It would appear that ths Type I antigen no longer serves its purpose
in the presence of the immune substances formed during convalescence, and


172         P~~eumococcnl Types
the pneumococcus consequently develops its Type II side. I have not so
far been able to find a Group IV st,rain in a case of pneumonia due to Type II
(in one instance a Type III strain was found in associat,ion with the Type II).
but my observations have not been sufficient,ly numerous to justify a con-
clusion on this point.
While these suggest,ions of a regular sequence of changes in the type of
pneumococcus before the development of pneumonia and during recovery
are necessarily tentative in character, they are in harmony with the experi-
mental data and amplify t'his line of t,hought.
The formation of a Group IV strain from a Type I might be considered
as an adaptation on the part of the Type I pneumococcus to the altered
conditions consequent on the development of immune bodies. These make
it difficult for the Type I to survive as such (i?t vitro the R form is the
response), and in assuming Group IV characters it makes some sacrifice of
its antigenic complexity and, with that, of infectivity in exchange for a
greater degree of resisting power to the animal tissues. Type III exhibits a
still greater instance of change in that direction, since it is very slightly
invasive but fatal in its effects once it is established in the body. It is more
difficult t,o produce protective sera in rabbits with Group IV and Type III
strains than with Type I, while Type II occupies an intermediate position.
In t,he int~eraction between the animal tissues and the bacterium one is
apt t,o consider the bacterium as playing a purely passive part and to over-
look the possibility that the various forms and types may be assumed by it
to meet alt,erations in its environment.
What: for instance, is the meaning of the change to the R form? Xost
writers have regarded it as a degenerative change due to unfavourable con-
ditions for growt,h. While this is true in a sense, since in the R form the
bacterium lacks certain important attributes characteristic of the S form,
there is some evidence of its being rather a vit'al adapt,ation, as I'. Hadleyl
has suggested.
In the case of t,he pneumococcus the change to the R form is brought
about most rapidly in immune serum which, nevertheless, provides an ex-
cellent medium for the growth of both the R and the S form. The effect of
the serum is due to the specific immune substances, and, as a result, the
pneumococcus becomes susceptible to phagocytic action. If, as is probable.
t,he immune bodies exercise a similar influence in Go during successful rc-
sistance? the animal has achieved its end in rendering the invader harmless.
By assuming the R form the pneumococcus has admitted defeat, but has
made such efforts as are possible to retain the potentiality to develop afresh
into a virulent organism. The immune substances do not apparently continue
to act on the pneumococcus after it has reached the R stage, and it is thus
able t,o preserve remnants of its important S antigens and with t,hem t,he
capacity to revert to the virulent form.
        1 7'Le JwruaZ of I~?fertio~r.s Disenws, 1927, 40, pp. l-312.


F. GRIFFITH

While the R form may be the final stage in the struggle of the bacterium
to preserve its individuality, I look upon the occurrence of the various sero-
logical races as evidence of similar efforts to contend against adverse circum-
stances. These are more successful in that the S form is retained and, in
addition, increased powers of resistance are acquired but at the sacrifice of
invasive properties.

The experiments on enhancement of virulence and transformation of type
   __. -..
suggest an explanation of the manner in which a pneumococcus residing as
-.
an apparently harmless saprophyte in the nasopharynx acquires disease-
--
pyp_duhg. powers. So long as it retains certain potentialities, indicated by
t,he possession of traces of S ant'igen, the most attenuated pneumococcus may
develop the full equipment of virulence.  The first essential is a situation in
which it can multiply, unchecked by the inhibitory action of a healthy
mucous membrane. In the nidus thus formed the pneumococcus gradually
builds up from material furnished by its disintegrating companions an anti-
genie structure with invasive properties sufficient to cope with the resistance
of its host.

When recovery from pneumonia takes place, the formation of immune
substances initiates the retrogressive changes in antigen structure resulting
in the production of the Group IV and Type III pneumococci which probably
have increased resisting powers but diminished capacity for invasion.

These considerations which relate to an individual case of pneumonia are
capable of application to an outbreak, of epidemic disease in a community.
Thus the consequences which ensue on the decline of an epidemic are not
only an increase in the number of insuscept,ible individuals but also an altera-
tion in the character of the infective organism.

IV. SUMMARY.

1. In the course of the examination of sputum from cases of lobar pneu-
monia, observations have been made on the incidence of the chief types of
pneumococci. In the district from which the material was obtained, there
was an apparent local diminution in the number of cases of lobar pneumonia
due to Type II; the figures were 32.6 per cent. of Type II cases in the period
1920-22: and only 7.4 per cent. in the period 1924-27. The incidence of
Type I was approximately the same in the two periods, the percentages being
30.6 and 34.3.

2. Several different serological varieties of pneumococci have been ob-
tained from the sputum of each of several cases of pneumonia examined at
various stages of the disease. This has occurred most frequently in cases of
pneumonia due to Type I, and in two instances four different types of
Group IV were found in zddit'ion to the chief types. The recovery of different
types is facilitated by t,he inoculation of the sputum (preserved in the re-
frigerat,or), together with prot'ective sera corresponding to the various types
in the order of their appearance.


174         Pneumocmcnl Types
3. TWO interesting strains of Group IV pneutnococci have been obtained
from pneumonic sputum.
One was an R strain which produced typical rough colonies, yet preserved
its virulence for mice and its capacity to form soluble substance. This R
pneumococcus developed a large capsule in the mice, which died of a chronic
t,ype of sept,icaemia. A strain producing smooth colonies was obtained from
it in the course of a prolonged series of passage experiments.
The second strain, which was proved not to be a mixture, agglutinated
specifically with the sera of two different types. In the peritoneal cavity of
the mouse the specific soluble substance of each type was produced.
4. A method of producing the S to R change t,hrough ageing of colonies
on chocolate blood medium containing horse serum is described. After two
to t,hree days' incubation small rough pataches appear in the margins of the
smooth colonies, and from these pure R strains can be isolated.
5. It has been shown that t,he R change is not equally advanced in the
descendants of virulent, pneumococci which have been exposed to the action
of homologous immune serum.  Some R strains form traces of soluble sub-
stance in the peritoneal cavity of the mouse; these revert readily to the
virulent S form and, in addition, are able t,o produce active immunity. Others
show no evidence of S antigen; spontaneous reversion takes place with diffi-
culty, if at all, and they are incapable of producing active immunity. The
stronger the immune serum used, the more permanent and complete is the
change to the R form.
6. Restoration of virulence to an attenuated R strain, with recovery of
t,he S form of colony and of the original serological type characters may be
obtained by passage t'hrough mice. The change from the R to the S form is
favoured by the inoculation of the R culture in large doses into the subcu-
taneous tissues; but the most certain method of procuring reversion is by
the inoculation of the R culture, subcutaneously into a mouse, together with
a la.rge dose of virulent culture of the same type killed by heat.    *

  Incubation of such a mixture in vitro does not induce reversion.
7. Reversion of an R strain to its S form may occasionally be brought
about by the simultaneous inoculation of virulent cult,ure of another type,
especially when this has been heated for only a short period to 60" C., e.g.
R Type II to its S form when inoculated with heated Type I culture.
8. Type I ant,igen appears to be more sensitive to exposure to heat than
Type II ant'igen, since the former loses the power to cause reversion when
heated to 80" C., whereas Type II culture remains effective even after steaming
at 100' C.
9. The antigens of certain Group IV strains appear to be closely related
to that of Type II, and are equally resistant to heat. Steamed cultures of
these Group IV strains cause the R form derived from Type II to revert to
its S form, while they fail to produce reversion of the R form derived from
Type I.


               F. GIUJFITII               175

  IO. The inoculation int.o the subcutaneous tissues of mice of an attenuated
R strain derived from one type, together with a large dose of virulent culture
of another type killed by heating to 60" C., has resulted in the formation of
a virulent S pneumococcus of the same type as that of the heated culture.
The newly formed S strain may remain localised at the seat of inocu-
lation, or it may disseminate and cause fatal septicaemia.
The S form of Type I has been produced from the R form of Type II,
and the R form of Type I has been transformed into the S form of Type II.
The clear mutinous colonies of Type III have been derived both from
the R form of Type I and from the R form of Type II, though they appear
to be produced more readily from the latter. The newly formed strains of
Type III have been of relatively low virulence, and have frequently remained
localised at the subcutaneous seat of inoculation.
Virulent strains of Types I and II have been obtained from an R strain
of Group IV.

11. Heated R cultures injected in large doses, together with small doses
of living R culture have never caused transformat,ion of type, and only rarely
produced a reversion of the R form of Type II to its virulent S form.
12. The results of the experiments on enhancement of virulence and on
transformation of type are discussed and their significance in regard to
questions of epidemiology is indicated.

(ICIS. received for publication 26. VIII. 1927.-Ed.)