STUDIES ON THE BIOLOGG OF STREI?TOCOCC
I.ANTIGENIC~LA~TI~N~HI~S BETWEENSTaAINs OF%REPTOC
                      EiEMoLYTICrJs.

By A. R. DOCHEZ, M.D., 0. T. A-YY, M.D., AND R. C. LANCEFIELD.   ;
(From the Zhs#tal of The Rockejd&v I&&& for Medical Research.    .

      (Received for publication, June 1, 1919.)  /
The importance of the problem of the systematic classification of

bacteria for the proper understanding and control of infectious
diseases is becoming increasingly evident.  Such study is necessary
not only in the elucidation of the biological relationship existing
between varieties of the same species of bacterium, but is also essential
to the working out of epidemiological problems and to the de&l:
opment of knowledge useful in the &ort to control infectious piseases
by means of specitic therapeutic and prophylactic measures. Bac-
teria closely resembling those responsible for the pathological process
in many acute .infections have been found to be present and to live,
apparently without harm to the host, on the mucous membranes of.
a large proportion of normal individuals. The resemblance of the
pathogenic to the harmless variety of microorganism is frequently
so close that in many instances tests of particular specificity are re-
quired to show the existing biological differences. In fact, the problem
in etiology .today is to determine not only the bacterial species cak-
ing a given disease, but in addition the number of varieties of the same
species that qe pathogenic, and whether common and important
non-pathogenic varieties exist.  The study is one of varying com-
plexity, and methods suitable to one species do not give the desired
information when applied to another.

The purpose of such studies may be broadly defined as an effortto
relate fixed and determfna ble characteristics of bacteria to patho-
genicity. Though fluctuating variations of bacteria probably occur,
it seems not unlikely that in most diseases a sutIiciently constant equi-
librium has been attained to justify the usefulness of the effort. .-The
                      179


180            BIOLOGY OF STREPTOCOCCUS. I

methods employed in these studies are numerous.  In some species
morphological and cultural characters give `important though some-
what limited information, and in many the biochemical reactions
are of great significance. The most serviceable method, however, for
obtaining the particular kind of knowledge desired `is the study of
immunological relationships. For some as yet rrnexplained reason
the latter specitic reactions are very constant among the pathogenic
varieties. In the following study of Streptococcus hamolyticus chief
dependence has been placed on the knowledge obtained from the
study of these immunity reactions.

In the various czhsifhtions of the streptococcus group as a qhoIethat have
been proposed, the custom has been to consider the strains that effect hemolysis
of red blood cells as constituting a single or unit type (1).  The validity of this
assumption has been questioned and there has been much study and discussion
of the probability of the existence of variations within this group, some evidence of

which has been obtained from the study of sugar fermentations (2).  Recent studies
(3) indicate that certafn broad lines of differentiation may be shown between
hemolytic streptococci of human origin, and those of bovine origin whether found
  in milk or cheese. Hemolytic streptococci of the human type are usually found
in association with some pathological process such as puerperal sepsis, septicemia,
erysipe-las, bronchopneumonia, or other conditions, and hemolytic activity is
generally considered as an indication of pathogenicity. With the development
of our knowledge (4), however, these organkns have been found with iucreasing
frequency when no pathological lesion has been apparent. Investigators for
many years have been interested in these strains, and dkcuasion has centered
about the unity or multiplicity of the group (5).  The evidence in general favors
the belief that hemolytic streptococci pathogenic for human beings comprise a
single type.. In the present paper it will be shown that by the use of properly
  controlled immune reactions dXerential characters between individual strains
&n be shown to exist.

During the winter of 191748 in the United States, there occurred
in numerous localities a great increase in the incidence of a previously
rather infrequent type of bronchopneumonial  The highest morbidity
rate and earliest appearance of this disease were in military can-
tonments. In the spring of 1918, however, the type of infection
under consideration was commonly observed in the civilian popu-
!lation. The disease first appeared as a secondary pneumonia fol-
lowing measles, but soon instances of apparently primary infection
.of the lungs were observed. Numerous studies of the bacteriology


A. R. DOCHEZ, 0. T. AVERY, AND IL C. LANCEFIELD     181

of this condition have demonstrated that the infectious agent re-
sponsible for the lung lesion was in almost every instance Strepto-
coccus hamolyticus. As a result of the widespread incidence of the
disease the latter organism became extensively distributed and was
frequently found as a secondary invader in acute lobar pneumonia,
and as a common inhabitant of the normal throat.

The material used in the present study was obtained from the
military establishments in the neighborhood of Fort Sam Houston,
Texas. The sources of the individual strains were the throats of
patients suffering from acute measles, the sputum of patients with
bronchopneumonia both primary and secondary to measles, patho-
logical material obtained from cases of bronchopneumonia and acute
lobar pneumonia, and the throats of healthy individuals who had
been directly or indirectly exposed to infection in a variety of ways.

All the strains of hemolytic streptococcus employed in this study
possess the typical characteristics of the group. They are hardy
organisms and grow readily in meat infusion broth and on blood agar
slants. They survive for many months when grown for 18 hours
in rabbit blood broth and subsequently placed at refrigerator tem-
perature.  In meat infusion broth the growth has been of two types
-a granular sediment with clear supernatant fluid and a flocculent
sediment with turbidity throughout the remainder of the tube.  All.
the organisms are strongly Gram-positive, grow in chains of varying.
length, and are bile-insoluble.  Capsule formation has not been ob-
served. On plates two types of colonies are seen-a small, round,
smooth colony and a moist ameboid colony with a slightly roughened
surface. AU the strains are actively hemolytic, showing a wide zone
of hemolysis on the surface of rabbit blood agar plates; hemolysis is
complete in 2 hours, when a 5 per cent suspension of washed rabbit
blood cells suspended in salt solution is mixed with an equal quantity
of a 24 hour broth culture. The power of the tierent strains to
ferment the usual test substances for streptococcus has been studied.
The majority fall in the group of Streptowuxs pyogms according to
Holman's classification of streptococcus on a basis of sugar fermen-
tations. None of the strains ferments inulin and about 20 per cent
of these actively hemolytic strains possess the power of fermenting
mannite. The latter characteristic will be shown to have an inter-


182        BIOLOGY OF STREPTOCO-CCUS. `I

esting relationship to the immunological classitication of these
organisms.                       .
The virulence of this group of hemolytic streptococci is low for
the ordinary laboratory animals in comparison with such an or-
ganism, for instance, as pneumococcus.  Doses of 1 cc. or more of a
24 hour-broth culture administered intraperitoneally are required to
hill guinea pigs and rabbits. Furthermore, repeated passages through
these animals fail to bring about a considerable accession of `viruIence.
The fatal dose for white rats and mice is smaller, usually in the
neighborhood of 0.1 cc. of a broth culture. By continuous passage
through rats and mice it has been possible to raise the virulence of a
certain number of strains to a point where 0.001 cc. of broth culture
is lethal for a rat in 24 hours and O.OOO$MjOl cc. for a white mouse.
On the other hand, many strains cannot be raised to this high degree
of virulence even after the most persevering eiXort.  Once the max-
imum of virulence is attained, this quality persists without renewed
animal passages for an indeCnite period of, time.

The sources of the strains of Streptococcus hamdytkus studied and
some of their common characteristics are shown in Table I.

Although the finer differential classification of single species of
bacteria by means of immune reactions is still in the earlier stages of
development, enough evidence has been gathered to indicate that
the more highly parasitic varieties of the species are more likely to
consist of a limited number of unit types than are the less parasitic
or the saprophytic members.  In other words, unity of type seems to
characterize the disease-producing microorganisms, whereas hetero-
geneity is more common among the non-pathogens. If this'assump-
tion is true it then becomes important to choose for purposes of
classiiication the immune reactions which bring out most sharply
the kind of differences sought, rather than a reaction which develops
the basic relationship existing between all strains of the same species.
For this purpose we regard the reactions of agglutination and pro-
tection as of superior usefulness to those of precipitation and com-
plement fixation. The validity of any final classification arrived at
depends, of course, upon the possibility of fitting accurately into
such a classitication a large number of strains freshly obtained from
their natural environment.


A. R. DOCHEZ, 0. T. AVERY, AND R. C. LANCEFIELD     183

                       TABLE I.
soz4rce and co?nmo?l Chamcters of Strains of Stre~t~c0ccu.s hmwlyticus Studi4d.

"Es?      soorce.           clini diagno8ie.      Hcmolysia.

s29
s 114
s 118
S 124
SIB5
SM.6
Sl49
s 151

Sputum.
  "
FleuraI fluid.
sputum.
Throat.
chest fluid.
Blood.
PIeuraI fluid.

s2
s3
s 14
s 111

Autopsy 0.
" yd.
  "

-Chest fluid.

S53
564
SC54

S80
s140

s II
S 16
s31
S44
SS3
s95
s 125
Sl44

Ttma+
  u
  `1


  `4
  `L


Sputum.
  "
Throat.
  `6
  `I
  "
  `,
  `I


  `#
  I`


  "

Types3.

          following ++++++---
measles.
    "         u      ++++++---
    `8         `6      ++++++---
    "         "      ++++++---
    "         "      ++++++---
    "         "      ++++++---
    u         u      ++++++---
    "         U      ++++++---
    44      "      ++++++---

Bronchopneumonia.
      "
      "
      "

++++++---
++++++---
++++++---
++++++---

MePsla.        ++++++--- .
   64            ++++++---
   `&               ++++++---
Germanmeades.     ++++++---
  "      "           ++++++---

Lobarpn~llia(TypeI).     ++++++---
"     u           ++++++---
"      "           ++++++---
"      `4            ++++++---
46      "           ++++++---
,*      "           ++++++---
"      u           ++++++---
4`      6`           ++++++---

Rmmonia.             ++++++---
   "           ++++++---

Incipient turn       ++++++---


184          BIOLOGY OF STBJWTOCOCCUS. I .


              TABLE I-Continued.

Type S 23.

S39

folIowing

s 78
s 101
s 107

Pleural fluid.
  4t 1`
sputum.

Bronchopneumonia
measles.
      `6
      "
      "

"
"
"

s 27
S 67
s 120

Autopsy (blood).
Blood.
Autopsy (blood).

Bronchopneumonia.
      "
      "

S98
S 116
s 117

Tbruat.
"
`6

MC.&=.
  1`
  1`

s9
S 23
S 56
S6S
s 7s
s 133

1`
1`
Autopsy (lung).
sputum.
Throat.
"

Lobar pneumonia.
6`      "
`I      l`
S`      `4
`6      "
"      "

s104
s 130


S46
s 122

Pneumonia.
  "

folIowing ++++ + + - -
      ++++++--

"      ++++++--
      ++++++--
"      ++++++--
      ++++++--
"      ++++++--
      ++++++--

      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--

      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--

      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--
      ++++++--
      +++-t++--
      +++-t++--

      ++++++--
      ++++++--
      ++++++--
      ++++++--

      ++++++--
      ++++++--
      ++++-i-+--
      ++++-i-+--

-










.
.


.
.



.
.
.
.
.




.

Incipient tuberculosis.
  6`      "

-


A. R. DOCJiJZZ, 0. T. AVERY, AND R. C. LANCEFIELD    185

TABLE I-continwd.

Sf37

S6
s 35
s55

s 10
s37
S43
S60
S66
s 71
s 86
s 88
S 89
Sloe
s109
S 123
S 127
S 128

s4

s 19
s 62
S 72

s 21
s 141
s IX

S 265

s26s

Pkual fluid.

AU~WJY 0.
Plealralfhid.
sputum.

Throat.
id
  "
  4`
  II
  u
  "
  "
  `4
  `6
  "
  Id
  "
  4`

Foot. .

Blood.

   Type s 60.

Bronchopneumoti   folloming
measles

Bronchopneumonk
      "
      `4

Measles.
  d`
  "
  "
  "
  "
"
  "
  "
  "
  4`
  u
  u
  "

Germanmeasles.

Cibar pneumonia.
6`     `4
"     "

blcipient tuberculosis.
  1`      6`
  "      "


CelbllitiS.

Erysipelss.

+++++++--


+++++++--
+++++++--
t++++++--

+++++++-;
++++-f-++--
+++++++--
++++++-l---
+++++++--
+++++++--
+i-+++++--
t++++++--
t++++++--
t++++++--
t++++++--
t++++++--
t++++++--.
t++++++--

t++++++--

t++++++--
+++++++--
t++++++--

t++++++--
t++++++--
t++++++--

+++++++--

+++++++--


186          BIOLOGY OF STREPTOCOCCUS. I

             TABLE 1-&?&d.

Sl

AUWSY Wd.

s20
s 110
S 138

" wad).
sputum.
  `8

SSO
S84
s 139

Pleural fluid.
  " `6

Throat.

s 11s

"

s 15

6`

S32  Autopsy Ouw$.

559
s93
s97
S 136
S 142

  `,   (blood).
sputum.
Pleural fluid.
Autopsy Owd.
sputum.

S49

`1

s 24

S 18
S 26
S42
s 51

  s63
  S%
  s 102
  SlOt
  s 1oe
  s 142

Throat.
4`
"
  "
"
"
  "
  `6
  u
  I`

-

-
Bronchopneumonia   foBowing
measles.
      "         u
      u         u
      "         *<
      "         u
      "         `I

Bronchopneumonia
Germanmeasles.

following

Measles.
  `1
  "
  6`
  "
  "
  "
  ,`
  "

"

-

TypeS84.

Bronchopneumonia   following ++++ + + -
measles.
      I`         u      ++++++-
      u         "     +-l--i-+++-
      "         u      ++++++-

Bronchopneumonia.        ++++-b+-
      `4             ++++++-
      "             ++++++-

German measles.          ++++++-

Lobar pneumonia.         ++++++-

    UnclassZed.

.
.


.
. .
, .


. .


. .

--


--
--
--


--
--
--


--


--
-

++++++---

+i-++++---
++++++---
++++++---
++++++---
++++++---

+++++.-i----


++++ +:-k - - -

+++++-l----
+-t+++-i----
+-t++++---
++ ++--+
++++*+---
++++++---
++++++---
++++++---
++++*+---
++++++---


A. R. DOCEJX, 0. T. AVEBY, AND R. C. LANCEFIELD     187

s 17
s47
S48
S54
S68

S69
S 87
S90
S99

S34
S36
s 121
s129
s 155


s264

S 271


S 272


S 273


S 276


S 277

s286


s288

Throat.
6`
"
sputum.
Throat.

Autopsy (lung).
sputum.
Thmat.
sputum.

Throat.
u
u
(I
64

B&d.


u


Pus.


6C


u


4` .(abdumen).

Pleural fluid.

sputum.

Gwmanmeasles.
  "     `C
  u     "
  "     "
  "     "


Lobar pneumonia.
"      `L
"     "`
"      "

Iucipimt tu~osis.
"      `L
II      u
6`      `6
U      u

Osteomyelitis.

septicemia.

Abscess (mea&s).

scarlet few+

Pelvic abscess.


Peritonitis.

Pneumonia.


    U*

+   -- +--
+++ +----
t+++++---
t+++++---
t+++++---

t+++++---
t+++++---
t++i++---
t+++++---

t++++++-+
t+++++---
t++++----
t+++*++--
t+++++---

t+++++---
t+++++---.

t+++++---

t+++++---

t+++++---

t+++++---

t+++++---

t+++++---


188

BIOLOGY OF STREPTOCOCCUS. I

The Riaction of Agglutiization.

Specific agglutination has been found to be one of the most ser-
viceable immune reactions for purposes of the biological classif%
cation of bacteria. In the typhoid and pneumococcus groups, for
instance, it serves to distinguish clearly the difIerent varieties from
one another. It is likewise applicable to the classification of many
other microorganisms.  Efforts to classify the streptococci by means
of this reaction apparently have not illuminated materially the
relationship of one strain to another, nor have they shown a definite
relationship between certain strains and a particular pathological
process.  A number of explanations of this fact may be proposed.
In many instances no attention has been paid to the broader groupings
of streptococci as determined by hemotoxin production and the fer-
mentation of test sugars.  Also streptococcus most frequently acts
as a secondary invader in the production of disease, and it is prob-
ably an unwarranted assumption to suppose that type specificity is
closely related to the character of the pathological process.  One of
the chief obstacles to the successful carrying out of the agglutination
reaction has been the tendency of all types of streptococcus to undergo
spontaneous granulation, and when used for tests to exhibit the
phenomenon of non-specifk agglutination. As a result of this, the
reactions must usually be read against a more or less granular back-
ground, making it dif3icult, if not impossible, to distinguish between
the non-specific and the specific influences.  The tendency to spon-
taneous clumping is occasioned by several factors, only a few of which
are understood.  For instance, a homogeneous suspension of a granular
streptococcus can easily be prepared by washing the organism several
times with distilled water, and then resuspending in the same medium.
The suspension will remain homogeneous for an indefkite period of
time. If sodium chloride in concentrations above 0.06 per cent is
added to the suspension, granulation immediately ensues. Many
other salts act in the same manner.  Substances antagonistic to this
salt action may be added to the medium and function even to the
extent of suspending the participation of the salt in the immune
reaction, so that specific agglutination may be completely inhibited.
Fortunately intermediate combinations can be found in which most


A. R. DOCEEZ, 0. T. AVERY, AND R. C. LANCEFIELD     189

strains remain diffuse and in which the salt is stiIl able to fuhilI its
part in the immune reaction.  The most useful substance of this
hind is ordinary meat infusion broth to which 1 per cent peptone has
been added.' In addition, if streptococci are exposed to too great
acidity the tendency to granulation is increased.  In order to avoid
this, the reaction of the medium may be so adjusted and such quan-
tities of balanced phosphate solutions added that during growth an
acidity greater than pH 7.1 is not attained. Certain other unde-
termined factors cause granulation, which may be defined as a general
unsuitability of the medium for growth, and these can be eliminated
only by experimenting with di.Berent preparations.

Technique.

The immune sera used in the agglutination and protection tests
were obtained by the immunization of rabbits, sheep, and dogs.  The
animals were inoculated intravenously with repeated doses of heat-
killed organisms, and in most instances a certain number of doses of
living organisms was given. The employment for immunization of
freshly isolated unpassed human strains, or the use of the same
strains after a series of animal passages, does not alter in any recog-
nizable way the specific qualities of the serum.  The agglutinin and
protective titer of the sera has remained undiminished for many
months after the time of bleeding.

Great care is taken in the preparation of the organisms, to be used
in the agglutination reaction.  The broth is made from carefully
selected meat, and instead of the usual sodium chloride a sticient
quantity of a balanced phosphate mixture is added to give the re-
quired salt concentration and to adjust the hydrogen ion concen-
tration at a pH of 7.4.  When Streptococcus hmnoZyticua is grown for
24 hours in a medium to which no sugar has been added, it does not
develop an acidity greater than pH 7.2, which is just above the point
at which the tendency to granulation appears. The organisms are
removed from the culture medium by centrifugalization and are

I The authors are greatly indebted to Dr. CharIes Krumwiede, Jr., of the
Research Laboratories of the Department of Health of the City of New York,
for many helpful sug&stions in this technique.


190           BIOLOGY OF STREPTOCOCCUS. I

washed once or twice in broth with a pH of 7.4 prepared in the manner
described above. They are then resuspended in the same medium in
approximately the concentration that is attained after 24 hours growth
and are ready for use in the reaction. The sera to be employed in
the tests are made up to the various concentrations by dilution with
the same broth used for suspending the bacteria. The reaction of
each specific serum is controlled by a complete series of like dilutions
of normal serum of the same animal. Equal quantities of serum
dilution and bacterial suspension are added to each tube and the tubes
are then placed at a temperature of 55oC. for 1 hour. A temperature
of 55oC. must be accurately maintained during the entire period
and the tests must be read after the lapse of 1 hour, since if they are

TABLE IL

Puwcr of Antktre~tocouus Stnm, Ty#e.S 3, to Agglutinak Tm Rcpres~w
           Straim of the Same Tyjw.
          1               Dilution.

Strain No.

s3


s 14


Sl49

595

S80


S 118

s 111


S140

s 16

S83

Types3
NormaL
Types3
Nod.
Types3
Normal.
Types3
NormaL
Types3
Normal.
Types3
Normal.
Types3
Normal.
Types3
Normal.
Types3
Normal.
TypeS3
Nod.

:: 2
  . .   2
-A-
+* +*
  - -
-+* +*
- -
+* +*
   * v:
z-+ ++
  - -
+* +*
- -
+* +*
- -
+* +*
- -
+* +*
- -
+* +*
- -
++ ++
- -

I  f
. .   .z
2--
t* +*
- -
t* +*
- -
t* +*
- -
t+ +t
- -
t* +*
- -
t* +*
- -
t* +*
- -
t* +*
- -
t* +*
- -
t+ +t
- -

-





: .


: -


: .


. .


: .


: .


: .


: .


: .


. .

/
5 333
t; ; : :
----
t*+*+* +
- - - -
t*+*+* +
- - - -
t* +* +* +*
- - -_ -
t+ ++ ++ +t
- - - -
t* +* +* +*
- - - -
t*+*+* +
- - - -
t* +* +* +*
- - - -
t* +* +* +*
- - - -
t* +* +* +*
- - - -
t++++* +
- - - -

-g
--

;h -


dz -


t* -

t* -
+ -.


* -


+ -

4-t -

+ -


* -


TABLE III.

T& of Cross-Agglutindh ~ivns of Antktreptococcus Serum, Tyfie S 3, with
&h.s of Streptocotcus myticw 01 Other Ty#s and with Unclussi&d
                    Slrains.

-

str$n

s3

S 23

s 107

S 67

S65

s 75

S 128

s 55

S60

S 267


s4


S84


Sl

SSO

s 20


s11.5


s 31


s108

S87

S 288


S42


s 121

=

s. hzoTy"L.

  s3

  S 23

  S23


  S 23


  S23


  S 23


  S60


  S60


  S60


  6560


  S60


  S84


  S84


  S84


  S84


  S84


Undassibed.


   "

I`



u


"


`<

SCram.

Types3
Nod
Types3
Normal.
Types.3
NormaL
Types3
Normal.
Types3
Normal.
Types3
NotmaL
Types3
Nod.
Types3
Normal.
Types3
Normal.
Types3
Normal.
Types3
Normal.
Types3
Normal.
TYPeS3
NormaL
Types3
Normal.
Types3
Normal.
Types3
Normal.
Types3
Nod
Types3
Nomd.
Types3
NormaL
Types3
Normal.
Types3
Notmal.
Types3
Normal.

I

I

+









+

-

s
f;
-

-*

-'



+
-









*

191

+

*


-

+

-

Dilution.

+

-

-
3
f;
-

-A






-









-


-

-

-


192           BIOLOGY OF STRJiZTOCOCC!US. I

allowed to continue longer, non-specific granulation occurs.  If clump-
ing deveIops in the broth controls or in more than the Grst two or
three dilutions of normal serum, the reaction should be regarded as
unsatisfactory and discarded. By the use of this technique, it has
been possible to carry out reliable agglutination tests of various strains
of StrGptowccus hamdyticm and to show that constant type rela-

TABLE IV.

Power of AdistreQtowccus Serum, TyQe S 23, to Agglutinde Ten ReQresentdiva
           Strati of the Same Type.

Strain No.

S 23


s 107


S 27


s 39


S 56


S67


S98


s 101


s104


s 130

serum.

Types23
Normal.
Type S 23
Normal.
Type S 23
Normal.
Types23
Normal.
Types23
Normal.
Types 23
Normal.
Types23
Normal.
Types23
Normal.
Types23
Normal.
Types23
NormaL

-

8
1;
-
7-t

7-t

+

+
-.
+

+


-*


-*


+


-*


-

-



-.
_


. .









: .


: .






: .

-

s
.z
-
t-i

i-t

t-l

+

+

+

t-t
-
t-t

4-S
-
t*


-

-

-_
-_


- _


-_









_ _


_ _


- _


: -


-

Dilution.

   0
s     no o
   1 ml   a
I; 1; .. :
--A-
t+ ++ ++ +i
- - - -
t+ ++ ++ +t
- - - -
t++++* +
- - - -
t*++-i-* +
- - - -
t*+*+* +
- - - -
t*+* + +
- - - -
t+ ++ ++ +*
- - - -
t+ ++ ++ s't
- - - -
t+ ++ ++ +-t
- - - -
t*+++++*
- - - 7

q
;'
-
+


r*


*


*


*


*


-*


-*


-*


+


-

f
N
2
-



+









+

+

+

+


-

a
3
. .
1.




*









*


-


+


-

tionships exist, and that the types are sharply differentiated from
one another. The results of the application of the method to strains
of streptococcus described above are shown in Tables II to IX.

In these tables are presented the agglutination reactions of a certain
proportion of the total number of strains of Streptococczcs hmdyticzcs
tested. An analysis of the results shows that in the collection of


TABLE V.

Test of Cross-Agghti?tation Reacths of AntistreQtococcus Set%m, TyQe S 23, WUJ
   Strains of StreQtowccut ho%aolyticus of 0th TyQw and tith

S 23


s95

s 149

S80

s146

s 125


s 55


S60


s4


s 19


S6


s 20


s 50


S84


s 115


Sl


s 15


S 116


s49


St59


s 155


S 18


S99


S 24

  S 23


  s3


  s3


  s3


  s3


  s3


  S60


  S60

  S60


  S60


  S60


  S&L


  S84


  S84


  S84


  S84


unclassified.


   "

1,


"


"


"


"


"

SWUOI.

Types23
Normal.
Type S 23
$223
Normal.
Types23
Normal.
Types23
Normal.
Types23
Normal.
Types23
Normal.
Types23
Normal.
Type S 23
Normal.
Type S 23
Normal.
Types23
Normal
Types23
Normal
Types23
NormaL
Types23
NormaL
Types23
Normal.
Types23
NormaL
Type S 23
NormaL
Type S 23
NormaL
Type S 23
Nomal.
Types23
Normal
Types=
Normal.
Type S 23
Normal.
Types23
NormaL
Types23
Normal.

-


a
2
-

*









-

-


SI
5;
-
+

-

193

-


P
;
-
b*

-

-

-
P
3
;
-

-A









-.

-
%
2
;
-
t

- ,

-


194        BIOL?GY OF STBEpToCOCCUS. ' I

organisms studied it has been possible to detect four Merent types
of Streptococcus hux&yt&~. These types have been noted as Types
S 3, S 23, S 60, and S 84, from the number of the chosen represen-
tative. In addition to the type strains,' there remains a residue of
unclassified organisms. The summary for the total number of strains
studied is given in Table X.

TABLEi VI.

Power of AntiskGptoGoGcvs Sewn, TyQe S 60, to AggW Ten Reprcsmiatioc
           3rais.s of the Same Type.

Strain No.               %, .c   0   8
                          a N.  % ::
                               2 v) 'I
                                 t; 1   t; 2   ; 1;
                                 --  -- .- --

S60      WSm   ++ ++ +t    +t    +* i-* +
          NormaL      - - -                - -
s269     Types60   +* +* +* t* +  + *
          Normal.          - -
S 267     Types60  +=k ++ ++  - -   - -
                        t++* t* +  +
          NormaL          - -   - -   - -
s 55      %Fsm  -I-`t ++ +t    +*      *   * *

S 128   gtz%o     - - -                - -
               ++ ++ +t    +t    +* : . t* +
         Nd      -.- -                - -
s 72      Types60   +* +*    t*    t* +  + *
         NormaL  c-t +T-            - -   - -
S43      Typesa            t*    t* +* : . t* +
         NormaL      - -             - -   - -
s123     TjpeS60   ++ ++    t+    t++* : . 4-k +
        NormaL      - -             - Y   - -
S66      Typesa  ++++    t+    t* +A  + +
        NormaL I  - -             - -   - -
S 127             ++ ++ I I   t+    t+ +* : . t* +
                      - -             - -   - -
                                            -

Dihltion.

The total number of strains of Streptococcus hmnolyticw studied
was 125. Of these, 85, or 68 per cent, are comprised in the types
mentioned above, and 40, or 32 per cent, remain unclassified.  Work
with the unclassified strains is being continued and the indications
are that a certain number of other types will be discovered.  In fact,
two new types have already been encountered, one comprising five


TABLE VII.

rest of Cross-Agglutination Redions of Antistreptococcus Serum, Type S 60, with
   Struins of Streptococcus h@mdyticw of Other Types and with
              Urula.Wi&d Strains.

S60


s3

s 111

s146

S 80


s 14


S 23

s 78


S 98

s 107


s 122


S84

s 115

s 15

Sl

s 20

SW

S%

S2M

s47


s 10:

S48

s17

-
  S60


  s3


  s3


  s3


  s3


  s3


  S 23


  S 23


  S 23


  S 23


  S 23


  S84


  St34


  S84


  S84


  S84
l&h&d.


    "

"


"


`6


u


6`

s-.

Types60
Normal.
Types60
NormaL
Typesa
Nod
Types60
NomuL
Types60
Nod.
Types60
Normal.
Types60
Normal.
Typesa
Normal.
Typesa
Normal
Typesa
NormaL
Types60
NormaL
Typesa
NormaL
Types60
NormaL
Types60
Nod
Types60
Normal.
Typesa
Nod
Types60
Normal.
Typesa
Nomd.
Types60
Nomd.
Types60
Nod
Types60
NormaL
Types60
Normal.
Typed 60
NormaL

-


z
s
-
--t
-.









f
*









*
-






+

-I-


*


l-2






*

-


s
t;
-
--t









*

*


-
-
-






*


*


+

+

-


s
;
-
--I









-

-

3
;
-

.*









i

-









-
-








*





+

DilUthl.

-

5
2
-

-A


-
-









-

-
-









*









-

-









*









-

-
$
@!.
I
:
-

*









-

-




-
-




-









-

-
::
x
;'
-

*

-

-
w
a
;;
-

-

-
i
E9
-

-


- .









-

-

195


196         BIOLOGY OF STREPTOCOCCIJS. I

strains and another four strains, the immune reactions of which have
not as yet been completed.

The antistreptococcus sera used in the agglutination reaction were
obtained in the main by the immunization of sheep, a species of animal
which yields a highly specific agglutinating serum for Slre~`&cocclcs
hceneolyticus. Agglutination occurred in all the type sera in dilutions
of 1 : 1,000 or higher, with the exception of Type S 84, of which the

TABLE VIII.

Poww of Antistreptococcus Serum, Type S 84, to AggZutinute Eight Represetztutive
           Strains of the Same Type.

Strain No.

S&4


Sl


S.50


s 20


s 15


s 115

$139

S 138

S-.

Types84
Normal.
Types84
Normal.
Types84
Normal.
Type.584
Normal.
Type S 84
Normal.
Types&Q
Normal.
Types84
Normal.
Types84
Normal.

=
I

-


w
s
-
t*

t*

t*

t+


+

t*

t*

t*


-

$ i3
2 ;
--
  +*

i-t +*
- -
t* +*
- -
t+ +*
- -
t* +*
- -
t* +*
- -
t* +*
- -
t* +*
- -

-




.-









i -


-


.


: -

DilUtiOlt.

0
2  E
z ;
--
+ -t
- -
t* +*
- -
t* +*
- -
t* *
- -
t* +
- -
t* *
- -
t* +
- -
t* +
a -

-




-









-

-

0
a
;;
-

*
-
Et


+









-

-
3
N.
c.i
-

-

-

-

-
2
p1
-

-

agglutination titer has been consistently lower, usually not above
1: 320. The agglutination titer of all the type sera for each strain of
the same type has been approximately equal to the titer for the or-
ganism used for purposes of immunization.  There has been strikingly
little cross-agglutination between serum of one type and strains
belonging to another. The same lack of crossing is observed among
the unclassiIied strains with the few eXceptions in which certain of
these strains have shown some degree of agglutination in the type


TABIJZ IX.

Test of Cross-Agghthatioion hadon.~ of AktistrefltococclLs Serum, Type S 84, with
   Strains of Streptococcus holytinrs of Other Types ami with

Urulustifid Strains.

S84


s3


s 14


S80


5146


s95

S 23

s 107


s 122


s 78


S98


S60


S 128


s 19


s 141


s4

S 108
Sd7

SS4
s34 ~
S 106 ~

Type of
S. hanwlyticUs.

  S84


  s3


  s3


  s3


  s3


  s3


  S 23


  S23


  S 23


  S 23


  s 23


  S60


  S60


  S60


  560


  S60

Undassified.

   "

Serum.

Types84
Normal.
Types84
Normal.
Types84
Normal.
Types84
Normal.
Types84
Normal.
Types84
Normal.
Types&Q
Normal.
Types84
Normal.
Types84
Normal.
Types84
NormaL
TypeS8-4
Normal.
Types84
Normal.
Types84
Normal.
TypeS84
NormaL
Types84
Normal.
Types84
NormaL
Typesa
NormaL
-S&4
EM
Normal.
Types&Q
Normal.
Types84
Normal.

-


::
.s
-
I-=









*


*
*
*









*
r;
*
;'
+
3
T
+









-
197

-


E2
;
-

I-;









-

*
z
*
*


*
*
*
*

-

Q
z?l
5;
-

-t









-
-









*
T
*

t


-

-
si
2
;
-
+

-

-

-
w
a
3
-

-

-
$
p1
-

-'






i

-'



-


198           BIOLOGY OF STREPTOCOCCUS. I

sera, but not in su&.iently high dilutions to justify their inclusion
within the types. These facts show that it is possible by a series of
carefully conducted agglutination experiments to determine speciiic
type relationships between strains of Streptococcus hmnoZyt;czcS and
to show that the different types are immunologically distinct from
one another. The clearness of the agglutination reactions presented
is somewhat deceptive as to the ease and simplicity of the test.  It
must be remembered that Skeptococcjcs humolyticus is notoriously

         TABLE X.
Sumnurry of Agglutinutim Reactions.

No. of stmfas.

  s3.................................................. 29
  S23.......;......................................... 20
  s60................................................. 27
. s&l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     9

uIldassised.......................................... 40
Total typed.......................................... 85
                                           -
"  stlains studied.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    125

I   Percent.

21.6
7.2

32.0
68.0

variable in its reactions, and that very slight and indeterminable
changes in technique frequently obliterate almost completely the
specificity of the agglutination reaction.  In addition, a considerable
number of strains is invincibly granular under all conditions and
cannot be used, and occasionally strains are encountered which may
occupy intermediate positions, the exact understanding of which
needs a technique for the conduction of absorption experiments.

The Reactiun of Protection.

Study of the power of antistreptococcus serum to protect animals
against experimental infection with this organism has given rise to
a number of different points of view, both regarding its action against
strains from different sources, and against the same strain before and
after animal passage, and also concerning the kind and the different


A. R. DO-Z, 0. T. AVERY, AND B. C. LANCEFIELJI     199

effect of varying antigens used in the process of immunization.  For
a full discussion of these matters the reader is referred to the general
articles on streptococcus and to the. more important papers dealing
with these particular points (6). In this work they will only be con-
sidered where they have a particular bearing upon the subject under
investigation. Although in the present paper the classification of
Streptococcw hremoZyticzcs by means of the agglutination reaction has
been presented first, practically we have obtained our primary indi-
cations of the degree of antigenic dXerences between strains by means
of the reaction of protection.  Later each reaction has been used to
confirm the information obtained by means of the other.

In the successful carrying out of protection experiments two points
are of especial importance: first, the production of a serum of high
potency; and second, the possibility of raising the virulence of the
test strains of streptococcus to such a point that very minute doses of
culture are suf.Ecient to kill white mice in a limited period of time.
We have been able to produce sera in the manner alluded to above of
such potency that 0.5 cc. administered intraperitoneally is sufficient
to protect a white mouse against 100,000 lethal doses of a highly
virulent streptococcus. In order to produce such a serum many
animals must be used, only a few of which may give the desired
result. It has been possible to raise the virulence of many strains
by continuous passage through white mice and rats to such a point
that doses of from 0.000001 to 0.00000001 cc. of broth culture are
suflicient to kill the former animals in from 24 to 48 hours.  These
seemingly difficult conditions must be attained in order that &Ii-
ciently long-range protective titers may be carried out to insure the
reliability of the information obtained. Protection against one or
two lethal doses of a series of strains of streptococcus by a mono-
valent serum is subject to so many interpretations that the evidence
gained cannot be considered of much value in judging accurately the
antigenic relationship of the different strains.

The technique observed in the protocols given below has been as
follows: The potency of all sera has been titrated for the homologous
strain of organism and only the sera which gave a wide range of pro-
tection have been used.  For infection, virulent streptococcihave been
used which have been grown for approximately 18 hours in either


200             BIOLOGY OF STRJ3PTOCOCCUS. I

plain broth or ascites broth.  In the inoculation of animals the
technique advised by Neufeld (5) has been followed with only a
minor variation. The test animals have been injected intraperi-
toneally with 0.5 cc. of serum 24 hours before the conduction of the
experiment.  Tentative trials have shown that if the serum is given
simultaneously with the infecting dose, no protection results, and that
to insure success the serum must be given at least 8 hours before
infection. On the following day a series of virulence controls is
inoculated intraperitoneally, and the serum z&m& are injected in the
same manner with doses of cultures ranging from 0.001 to 0.00000001
cc. of broth culture.  Animals surviving for a period of 5 days are
considered to be adequately protected.  By the use of this method,
it has been possible to test the antigenic relationship of a considerable
number of virulent strains of Stre$&xucczls hmwlyticus, and the results
of these tests are set forth in the following protocols.

Protocol 1.

In this protocol is shown the titration of the serum of a sheep immunized against
Strain S 23.  The culture employed for infection was an 18 hour broth culture
of No. S 23, which had received eighteen passages through white rats and mice.
Each mouse had received 0.5 cc. of immune serum 24 hours previous to infection.

      vfirulence controls.              Protective power of Serum S 23.

Dose of culture.         Result.         Dose of culture.         Result.

   cc. .                          CC.
                          0.001         S.


0.00001     D.* i, 24 hrs.      .O.oool       D.in4days.
                         O.ooooOl       S.
O.ooooOl         I`  " 24. "       O.oooooOl        `<

O.oooooOl         "  6I 24 u      O.OOOOOOl        `L

*In the tables D. indicates died, S. survived.


Protocol 2.

In this protocol is shown the protective power of Immune Serum S 3 for two virulent strains of the homologous type,
for two strains of each of the heterologous types, and for two unclassified strains.  The technique was the same as that in

the previous protocol.

=
I

Dose of culture.

D.ooooOoo1 cc.   ?
       P

Streptocwcus No.    5w of
           atreptoLnccu9.   Type of serum.
                                  0.001 cc.     o.ooo1 cc.    0.00001 cc.    0.000001 cc.   o.oooooO1 cc.

s 3.w*       s3        s3      D. 14 hrs.     S.       S.        S.        S.
                   No serum.                           D. 20 hrs.   D. 23 hrs.

s 149.18      s3        s3        S.       S.        S.        S.
                    No serum.           D. 24 hrs.   D. 36 hrs.   D. 24 Ins.

s 39.3       S 23     s3   D. 16 hrs.   " 16 "    " 7 "   L` 19 "
                   No sentm.           " 16 "    " 16 I`    " 21 "

S 6t.Z       S 23       s3      D. 8 hrs.   `L 8 "    " 17 "    " 1, "   D. 19 hrs.
                    No serum.                           u 23 u    " 21 I`

S 128.14'      S60       s3     D. 24 hrs.   D. 24 hrs.   D. 48 hrs.   61 24 u
                   No serum.           u 24 Lt    " 36 `1    " 36 I`

s 60.106.      S60        s3      D. 18 lirs.   " 36 "    " 36 I`    " 20 "
                    No serum.           " 18 "      S.      6` 24 u

S 1.8'        S84       s3      D. 22 hrs.   " 22 "   D. 22 hrs.   " 22 "
                    No serum.           " 21 6`    " 21 "    L` 21 "

s 84.17'       S84       s3             " 13 1`   `1 19 "    " 19 "      S.
                No senn+                          " 18 "   D. 21 hn.
S 24.25'    UllClaSSilid.     s3      D. 14 hrs.  D. 16hrs.   D. 19 hrs.   " 31 "    `1 19 L`
                    No serum.                           `I 21 "    " 28 "

s 266-e         "        s3      D. 16 hrs.   D. 16 hrs.   D. 19 hrs.   (` 21 !`
                    No serum.           `I 1, "    `, 21 "    u 24 u
* The integer indicates serial number of the culture, the decimal shows the number of animal passages, and the exponent
the number of transplants since the last animal passage,, . I

   E
     Q*
D. 20 hrs.
" 60 "


In this protocol is shown the protective power of antistreptococcus setllfh, Type S 23, against two strafns of the
homologous type, against two strains of each of the heterologous types, and against two unclassi6ed strains.  The technique
was the same as that employed in the previous protocols.

Streptococcus No.

S 23.181

s 107.12'

S 3.21'

S 80.7'

s 55.22'

s 60.10'

S 84.18'

s 50.4'

S 276.31'

S 24.31'

S23

  SW



  s 3



  s 3



  560



  S60



  S&L



  S&M



UndaSStied.



    `4

Type of rerwn.

S23
No sernm.

S 23
No serum.

S 23
No serum.

S 23
No serum.

S 23
No serum.

S 23
No serum;

S 23
No serum.

S 23
No serum.

S23
No serum.

S23
No serum.

=
I

0.001 cc.

D.llhrs.

S.

D. 18 hrs.

D. 18 hrs.

D.2Ohi-S.

D. 20 hrs.

D. 12 hrs.

D. 18 hrs.

D. 16 hrs.

D. 16 hrs.

o.ooo1ae.

S.

S.

D. 18 hrs.



D. 18 hrs.



D. 36 hrs.
" 21 "

" 20 "
u 3-j "

" 20 `L



D. 36 hrs.



D. 40 hn.
" 20 "

" 16 `6

Dose of culture.

O.owol cc.

  S.
D. 19 hrs.

`< 16 1`
u 24 46

" 18 "
`I 18 `I

" 64 "
S.

D. 20 hrs.
" 22 "

`6 24 dd
`L 20 "

" 18 `6
" 16 `4

`I 18 "
`I 18 6`

`C 66 6`
" 18 6`

I` 16 `I
4, 16 `I

o.oowo1 cc.

  S.
D. 24 hrs.

  S.
D. 24 hrs.

`I 36 "
`I 36 4`

" 96 "
" 36 `8

`I 30 L`
" 20 "

  S.
D. 36 hrs.

`t 36 "
`, 20 `I

(6 18  "
" 36 "

`I #-J `<
" 4(-J `4

D. 20 hrs.

O.oooooOl cc.

D. 2Ohrs..

  S.
D. 24 hrs.

" * "
" 36 "

  S.
D. 36 hrs.

D.18hrs. _
  S.

D. 18hm.
" 18 I`


Protorol 4.

In thii protocol is shown the protective power of antistreptococcus serum, Type S 60, against two strains of homologous
type, against two strains of each of the heterologous types, and against two unclassified strains. The technique was the
same as that employed in the previous protocols.

Streptocoam No.

s 6O.W

s 55.221



S 3.22



s 80.b

s 75.8



S 23.196

s g4.12'

s 50.9



S 24.23?

S 276.31'

S60

S60

s3

s 3

S23

S 23

  S84



  S84



Unclassified.



    `#

lype of saum.

S60
No serum.

S60
No serum.

S60
No serum.

S60
No serum.

S60
No serum.

S60
No serum.

sa
No serum.

S60
NOWNHL

S60
No serum.

S60
No serom:

0.001 cc.

D. 19 hrs.

S.

D. 18 hrs.

D. 18 hrs.

D. 18 hrs.

D. 18 hrs.

D. 18 hrs.

D. 18 hrs.

D. 17 Ins.

D. `9hrs.`

o.oim cc.

S.

s. '

D.18hrs. .

D. 18 hn.

D. 21 hrs.
66 23 u

" 18 U

D. 18 hrs.

" 18 `6

" 18 #`

D. 17hrs.

D. 20 hrs.
u 23 66

Dose of cultnre.

o.oOOo1 cc.

  S.
D. 17 hrs.

  S.
D. 23 hrs.

" 38 "
" 18 "

" 18 "
U 18 "

" 20 L`
" 17 "

`I 18 4`
U 18 "

" 18 U
I` 18 6`

" 18 `I
`C 18 "

" 17 `L
" 17 u

6;. 24  66
`t 48 "

o.ooooO1 cc.

  S.
D. 19 hn.

  S.
D. 16 hrs.

" ,jo "
" 18 "

" 22 "
" 18 `<

D. 23 hrs.

66 24 t*
" 60 I<

" 22 u
" 20 "

" 18 "
" 18 "

u 24 66
" 65 "

" 41 "
" 33 `4

O.oaoclool cc.

D. 24 hrs.
" 18  `L


$6 24  46
" 18  "

D.6Ohl.s.
I` 18  "

D. 18 hrs.

`4 18  "

D.31hrs.


  Protocol 5.

In this protocol is shbwn the protective power `of antistreptococcus serum, Type S 84, against two strains of the
honiologous type, against two strains of each of the heterologous types, and against two unclassified str&. The tech-
nique was the same as that employed `iti previou's protocols.                           s
                                                                           I&

,
Streptocoicus No.

S 84.16'

.s 20.51

S 3.16'


S 14.29

`S 23.161

s 107.7' '

s 128.142

`S io..loa

`. I

: I . .
:  s: 152: 5'
    

`S 266.5'

Type of
streptococcus.
        .

s 84

S 84

s 3

s 3

i3 23

S 23

s&l

S60

Unclassified.

Type of serum.

s 84
No serum.
s 84
No serum.

s 84
No serum.

S84
No serum.

s 84
No serum.

S84
No,serum. .

S84
No s&q.

  S84
No serum.

s 84
No strum.

SM
No serum.

0.001 cc.

S.

s.

D. 17 hrs.



D. 16 hrs.



D. 18 hrs.



D. 18 hrs.



D. 24 hrs.


i

D. 18 hrs.-



D. 20 hrs.



D. 15 hn.

0.0001 cc.

  S.
D. 16 hrs.

  s. *
D:30 hrs.

D. 18 hrs.
  S.

D. 18 hrs.

D. 24 hrs.

Dose of culture.

0.00001 cc.

  S.
D. 26 hrs.

  S.
D. 55 hrs.


`( 20  "

(I 3-J "

D. 33 hrs.
LL 22 (6

D. 29 hrs.
  S.

D. 23 hrs.
tc 3s 4c

     ,
D. 16 hrs.
tt Jo `L

D. 24 hrs.

r-l


A. R. DOCHXZ, 0. T. AVERY, AND R. C. LANaFIELD     205

  Consideration of the above protocols shows that the type rela-
tionships manifest from the aggIutination reactions have been sub-
stantiated by the evidence obtained from the protection tests.  As a
matter of fact, each reaction has been used to supplement the other,
the first clue as to `the position of an organism sometimes being ob- i
tained by protection and sometimes by agglutination. On the I
whole, we are inclined to place- greater confidence in the reaction of !
protection than in that of agglutination, and would be slow to draw 1
general conclusions concerning type specificity from agglutination /
alone with such a variable organism as streptococcus, unless the \
results of this test could be confirmed by some other specific reaction \
such as protection. The sera prepared, as is seen from the protocols, \
have afforded a high degree of protection to white mice against in- 1
fection with organisms of the homologous type. Little or no pro-

tection results when serum of one type is employed against organisms
of heterologous types.  There are, of course, some exceptions to this
general rule.  Occasionally strains of Streptococcus hamolyticus are
encountered against which all type sera afford a varying degree of
protection, and sometimes a serum is obtained from one strain which
will protect against an organism of another type, and when the re-
action is reciprocally reversed no protection results. `At present our
k,nowledge is insufficient to discuss these intermediate reactions in-
telligently, and their elucidation must await further development of
the technique.  In all it has been possible to raise the virulence of
31 strains to a point where protection experiments could be per-
formed.  Of these, 7 belonged to Type S 3,8 to Type S 23,6 to Type
S 60, 7 to Type S 84, and 3 to the unclassified group. In view of
the difficulty of raising the virulence of -the. organisms it has been
                                                                      
found. advantageous to perform the reaction in two ways: first, to     I
test a single monovalent serum against a number of strains; and
second, to test a number of sera prepared from strains of the same
type against a single virulent strain of that type.  In Tables XI to
XIV is shown a summary of: the total number of protection experi-    I
ments performed.                        4


206           BIOLOGY OF STREPTOCOCCUS. I

TABLE XI.

Summury of the Pro&&e Pmver of Antistreptococcus Serum, Type S 3, agaifwt

Stra&s of the Homologow and Heterologws Tj

ts.

Type of serum.

s3
s3
s3
s3
s3
s3
s3
s3
s3
s3
53
s3
s3
s3

s3
s3
53
s3
s3
s3
53

s3
53

s3
s3

s3
s3
s3
s3
53

Undassiiie.d.
   "
   "

stmfn of s. hcmolyl-
icl`s used for pmduc-
tion of serum.

S3 (RabbitI).
s 3 ( " 1).
S3 (Dog 1).
s 3 ( " 1).
s3 ( " 1).
s 3 ( " 1).
s 3 ( " 1).
s3 ( " 1).
S 111 (Rabbit 2)
S 118 ( "  3)
s 2 ( "  4)
Sll ( (a  5)
S29 ( "  6)
S 16 ( "  7)

s3 ( "  1)
s3 ( "  1)
S3 (Dog 1).
s 3 ( `l 1).
s3 ( " 1).
s 3 ( " 1).
s 3 ( " 2).

s 3 ( " 1).
s 3 ( " 1).

S 3  (Rabbit 1)
s 3 ( "  .l)

s 3 ( " 1)
s 3 ( " 1).
s 3 ( " 1).
S3 (Dogl).
s 3 ( " 1).

S 24 (Rabbit 8).
S 24 (Sheep 2).
S 276 (Rabbit 9)

s3 (Type s31. o.ooooooO
s 14 ( " S3).   O.OooOOl
s3 ( " S3). o.ooooooO
s95 ( " S3).   0.00001
s80 ( " S3). O.OoOOol
s 149 ( " S3).   o.ooOOO1
s 146 ( " S3).   o.OOOOO1
s 144( " S3). O.aOOOl
53 ( " S3). O.OOoOOOl
s3 ( " S3).   O.OOOoOOl
s3 ( " S3). O.OoOOOOl
s3 ( " S3). 0.0000001
s3 ( " S3). o.alooLmo
s3 ( " S3). o.ooooooO

s 107 ( "  S23). O.OOOOOOl
S 23 ( "  S23). O.OOCMOl
S27 ( "  S23). 0.000001
S 67 ( "  S23). 0.0000000.
S 39 ( " S23). 0.000001
s 75 ( "  S23). O.OOOOOl
S 56 ( " S23). O.OOOOO1

s 128 ( "  S60). o.Ooom
s60 ( "  S60). O.OOoOol

s84 ( "  S84). o.ooooooo:
Sl ("  S&Q). o.OOOoO1

S 24 (unclassitied). O.OOOOOOO:
S 276 (   " 1. O.OooOOOl
S61 (   " 1. O.OOoOl
s 1.52 (   " I. O.OoOOOl
S266(   " 1. O.OOOOol


S 3  (Type S 3).   0.000001
s 14 ( " S3). o.OOoOOOO:
s 14 ( " s 3).   O.oOOOOl

ProOcp~&of
         .

s.0.0001 cc.
"0.001 "
" 0.001 `<
" 0.01      "
" 0.001 "
" 0.01      `1
" 0.01      `1
" 0.001     "

" 0.001     1`
"0.0001 "
" 0.001 (4
" 0.001 (`
" 0.0001 "
" o.Om (`

D.O.OOOOOOl "
" O.OoOOOOl `d
" O.OOml (a
" O.OOOOOOl u
" O.OOoOOl "
" 0.00001 "
`( o.OOoOO1 `(

" 0.000001 "
"O.OoOoOl "

" o.OoOOOO01 `<
" O.OOoOOl "

" 0 OoOooOOl "
s. 0:OaOOOO1 "
" 0.00001 "
D.0.OOOOO1 "
" O.OOOOOl "

`I O.OOOOOl I`
s. 0.00001 "
" o.oooOO1 " '


A. R. DOCBEZ, 0. T. AVERY, AND R. C. LANCEFIELD     207

TABLE XII.

Summary of the Protective Power of Antistreptococcus Serum, Type S 23, ugainst
     Strains of the Homologous ad Heterologous Types.

Type of serum.

S 23
S 23
S 23
S 23
S 23


S 23
S 23


S23
S 23
S 23


S23
S 23


S23
S.23

UIl&SS%ed.
   "
   "
   "
   "
   "
   `I
   `I
   "
   "
   ,`

S 23 (Sheep 1).
S 23 ( " 1).
S 23 ( " 1).
S 23 ( " 1).
S 23 ( " 1).

s 23 ( " I).
S 23 ( " 1).

S 23 ( " 1).
S 23 ( " 1).
S 23 ( " 1).

S 23 ( " 1).
S 23 ( " 1).

S23 ( " 1).
S 23 ( " 1).

S 24 ( " 2).
S24(" 2).
S 24 ( " 2).
S 24 ( " 2).
S 24 ( " 2).
S 276 (Rabbit 9)
S276 ( " 9)
S276( "` 9)
S276 ( " 9)
S 276 ( " 9)
S276 ( " 9)

S23 (TyyeS23). O.&l
s 107 ( "  S23). O.OOOOOOl
S27 ( "  S23). O.OOOOOl
s 75 ( (`  S23). 0.0000001
S 65 ( "  S23). O.OOOOOOl

s3 ( " S3).   O.coOOOol
S80 ( " S3).   o.OOoOm

s5.5 ( "  s.60). O.OOOOOl
s60 ( `(  $60). O.OOOOOl
S 128 ( "  S60). O.OOOOol

sso ( "  S84). O.OOOOOOl
s84 ( `L  S84). O.ooOOOl

S24 (unclassi6ed). O.OOOOOl
S276( "   >. O.OmOl

S27 (Type.523). O.&O001
556 ( "  S23). O.OOOOOl
s 107 ( "  S23). O.OOOOOl
s39 ( (`  S23). O.OOOOOOO
S 23 ( "  S23). 0.0000000
S27 ( "  S23). O.OOOWl
S56 ( "  S23). 0.00001
s 107 ( "  S23). O.OOOOOO1
s39 ( `(  S23). O.OOOOOl
S 23 ( " S23). 0.0000000
S67 ( "  S23). O.OOOOOl

Pmtective power of
0.5 cc. of serum.

s. 0.001 cc.
" 0.001 "
" 0.0001 "
" 0.001 "
" 0.001 "

D.O.OOOOOl "
s. O.o5oooOl L1

D. O.OOOOO1 "
s. o.cmOO1 "
D. O.OOOOOl "

(6 O.OOOOOOl u
" O.oLMOOOl "


" 0.00001 " .
" O.OOOOOl "

" O.OoOOOl "
" O.OOOOOl "
" O.ooooOl "
`( O.ooooOl ;'
1( o.OOOOm `(
" O.OOOOOl u
" O.OOoOol "
" o.OOOOOO01 `(
" O.OOOoOl "
" O.ooooooOl "
"0.OoOOO1 "


208           BIOLOGY OF STREPTOCOCCUS. I

TABLE XIII.

Sumnuy of the Protective Power of Antistreptococczcs Serum, Type S 60, against

Strains of the Homologous and %eterologous Ty

Type of serum.  Strain of S. hamolyt-
        icur used for produc-  Strain of S. ham++
           tion of serum.  and typeo~edfdf;,mfecttlon

  S60  S 128 (Rabdit 10).  S 128 (Type S 60).
  S60   S 128 ( " 10). S60 ( " S60).
  S60   S128 ( "  10). s 55 ( " S60).
  S60   S128( "  10). s4 ( " S60).
S&O   S 128 ( " 10). S72 ( " S60).
  S60   S 128 ( "  10). S 267 ( " S 60).


  S60   S 128 ( "  10). s3 ( " S3).
  S60   S 128 ( I` 10). S80 ( " S3).


  S60   S 128 ( "  10).  S75 ( " S23).
  S60   s 128 ( "  10). S65 ( " S23).
  S60   s 128 ( `(  10). S 23 ( " S 23).

  S60   S 128 ( `I  10). s84 ( " S&Q).
  S60   S 128 ( "  lo).' s 50 ( " S84).


  S60   S 128 ( "  10). S 24 (unclassified).
  560   s 128 ( u  10).  S276( " >.

Unclassified. S24 ( "   8).  S 128 (Type S 60).
   "    S276 ( "  9). S 128 ( " S 60).

Millild
e:hsl dose 01
S. hamolyt-
icus used fol
infection .

  cc.
O.ooOOl
O.OOoOOl
O.oOooOl
O.OOOOol
0.00001
O.oOOOOl


O.OOOOOO
O.OOOOOO


0300001
O.OooOOl
O.OOOOOO


O.OOoOOl
O.OOOOOO


O.OOOOOO
O.ooooOl

O.OoOOOl
O.OOOOOl

Is.

Protective power of
0.5 cc. of serum.

s. 0.01 cc.
" 0.0001 "
" 0.001 "
" 0.001 "
" 0.001 . "
" 0.001 "

D.O.OCOOOOl "
" O.OOOOOOl "

"0.OOoO1 "
s. 0.00001 "
D.O.OOOOOOl "

" O.OOOOOl " .
" O.OoOOOOl (`

" O.OoOoOl "
" o.ocxloo1 "

s. 0.001 "
" 0.0001 "


A. R.DOCHEZ, 0. T. AVERY, AND R. C.LANCJSFIELD     209

                      TABLE XIV.
Sum?lmry oj the Protective Power of Adstreptococcus Se&m, Type S 84, against
      Strains of the Homologous and %eterologous Types.

        strafn of s. /lmOlyc
Type of serum.  icur used for produc
           tion of s-.

S84
S&l
S84
S84
S84
S84
S84
S84
S84
S84


S84
S84


S84
S84
S84
S&l.
S84
S84
I-584


S84
S&l
S84


S84
S84
S84
S84
s84

S 84 (Sheep 3).
s 84 ( " 3).
s 84 ( " 3).
s 84 ( " 3).
s 84 ( " 3).
S84 ( " 3).
s 84 ( " 3).
S 1  (Rabbit 11)
s 1 ( " 11)
s 1 ( " 11)

S 84 (Sheep 3).
s 84 ( " 3).

s 8q ( " 3).
s 84 ( " 3).
s 84 ( u 3).
9 84 (  "  3).
s 84 ( " 3).
s 84 ( " 3).
S 1  (Rabbit 11)

s 1 ( `f 11)
S 84 (Sheep 3).
s 84 ( " 3).

s 84 ( " 3).
s 84 ( " 3).
s 84 ( " 3).
s 84 ( " 3).
s 84 ( " 3).

Unclassified. S 24 (Rabbit 8).
   `1    S 24 (Sheep 2).
   `1    S 276 (Rabbit 9).
.  `1     S 276 (  "  9).
   `1    S276 ( " 9).

                Minimal
Strain of S. hmwlyficus  lethal dose o
md type used for infection
    of mice.      S. kmoryt-
               icus used fa

infection.

                cc.
S84 (TypeS84). O.OOOOO1
Sl ("  S&Q). O.OOoOOl
s 20 ( " S&4), O.oouoOl
s50 ( "  S84). O.OOOOOl
s 139 ( "  S84). O.OOOOOl
s 110 ( `(  S84). o.OOOOOO1
s 15 ( "  SE%). O.OoOOOOl
Sl (`1  S&p). o.ooooooO
s84 ( (L  S84). o.ofxmoo
s20 ( "  S84). O.OOoOol

s3 ( " S3).
s 14 ( " S3).

S 23 ( " S 23).
S 107 ( "  S 23).
S39 ( " S 23).
S67 ( " S23).
S27 ( " S23).
S56 ( " S23).
S 107 ( "  S 23).

O.OOOOOl
o.ooooooO:


o.OOoOOO1
o.oOOOoO1
O.OOoOOl
0.000001
o.OOoOO1
0.00001
O.OOOOoOl

S 128 ( "  S60). O.OoOOOl
S 128 ( "  S60). O.OOoOOl
s60 ( "  S60). O.oOOOOl

S 276 (unMed). O.OOOOO1
S 277 (   " ). O.OOOOOl
S 152 (   " I. O.OOOOOl
S266(   " 1. O.OoOoOl
S24 (   " 1. oxMOO

S84 (Types&Q). 0.00001
Sl ($`  S&). O.OOOoOl
s84 ( "  S84). o.ooooooO1
s20 ( "  S84). o.OOOOO1
s.50 ( "  S&Q). O.OOOOOl

Protective power of
0.5 cc. of serum.

s. 0.001
`1 O.-JO05 :
" 0.001 "
" 0.0005 "
"0.001 "
" 0.001 "
" 0.001 "
" O.OOoOl "
" 0.0001 "
" 0.001 "

D.O.OOOOOl "
s. O.OOOOOl "

D.O.OOOOOOl "
s. O.OooOOOl "
" O.cQoool "
D.0.000001 "
" O.OoOOol "
" O.OoOOOl "
s. O.OOOoOOOl "

D.O.OOOOOl "
" O.OOOOOl "
"0.OOOoO1 "

" O.OOOOOl `(
s. O.OOOool "
D.O.OOOOOl "
"0.OOOoO1 "
" O.oooOol "

" o.OOOOo1 "
"0.OOOoo1 "
" o.oOOOOO01 (`
" O.OOooOl "
" O.OOoOOl "


210            BIOLOGY OF STREPTOcoC~ I

DISCUSSION.

The complete biological classification of any pathogenic micro-
organism presents a very complex problem.  The first phase of the
undertaking concerns itself with the development of reliable methods
for the determination of antigenic differences between members of
the species and the application of these methods to the discovery of
the immunological relationships between a limited number of strains
purposefully selected. In this way the degree of similarity and
diversity of type is shown and also the probable number of types, and
the proportion of classifiable to unclassitiable strains.  The next step
of necessity is the testing of the adequacy and universality of the in-
formation so gained by applying the tentative classification to a large
number of strains of the organism obtained under what may be
described as normal conditions of pathogenicity.  That some sort of
equilibrium has been established in nature among microorganisms
that have produced disease over long periods of time is not unlikely.
Indeed, evidence obtained from the study of pneumococci supports
this view (7), although departure from the norm may occur under
special conditions (8). After the relationships of the pathogens of the
species to one another have been discovered, it then becomes im-
portant for purposes of epidemiological study to compare by the same
methods the pathogenic with the saprophytic varieties. This task
requires years for its completion and many di&ulties and seemingly
unexplainable phenomena are encountered. In the beginning, the
broader lines of differentiation must be drawn, and divergent results
discarded for the time being, since, if the original conception is
correct, most of the discrepancies disappear with the advance of.
knowledge.

In this paper are presented the facts so far obtained in the present
study of Stre#ococcus hcemoZyt~us in accordance with the plan out-
lined above. The strains were collected in a limited community
during the course of what may be considered an epidemic of broncho-
pneumonia secondary to measles. Individuals, however, from all
parts of the United States were passing rapidly through this com-
munity which was a center for primary training of the aviation serv-
ice, so that a wider range of territory is represented than the im-


A.R. Docmz, 0.~. AVERY, m R. c. LANCEFIELD     211

mediate co~tmity itself. All the strains were investigated as to
their cultural reactions, bile solubility, capacity to hemolyze red blood
cells and to ferment the different test sugars, and as to hydrogen ion
concentration limiting their growth, and thus identified as accurately
as possible as Streptococcus hmolyticus of the human type.

A technique was then developed for studying the immunological
reactions of agglutination and protection. By the reaction of `agglu-
tination four distinct immunological types and a certain number of
unclassifiable strains have been discovered among the 125 strains
studied. Individuals of the same type are closely related to one
another immunologically, and the different types can be sharply
distinguished one from the other. In addition to the four types,
study of the reactions of which has been completed, there are in ad-
dition two other types, investigation of which is as yet incomplete.
The technique of the agglutination reaction demands great care,
both as regards the handling of the organisms and the preparation of
the medium for their growth.  In the medium used by us, a large
percentage of strains has grown sufficiently diffusely to permit the
preparation of stable suspensions. To what extent continuous growth
in this medium has promoted the tendency to diffuseness, and whether
the same percentage of freshly isolated strains will grow diffusely, we are
as yet unable to say. We have found that by the immunization of
sheep a highly specific agglutinating serum is obtained, but that the
serum produced from rabbits is not so specific and may show a wider
range of crossing, especially in one of the types of streptococcus de-
scribed. Variations in the specificity of di.lXerent animal sera have
been observed by students of the immunological reactions of menin-
gococcui. In order fully to understand this phenomenon, it would
be necessary to compare the specifkity of immune sera produced from
different species of animals by means of the method of absorption.
It is not as yet possible to undertake tbis hind of an investigation of
Streptococcers hmwlytkus.  The observation has been made, however,
that rabbit sera showing non-specifk cross-agglutination reactions in
general fail to manifest corresponding cross-protection reactions.

Whenever it has been possible to raise the animal virulence of
strains of Strept0coccu.s hamolyticus, the evidence obtained from the
agglutination tests has been confirmed by that- gained from the pro;


212            BIOLOGY OF STREPTOCOCCUS. I

tection reaction.  In all instances in which this has been done, one
reaction has corroborated the Endings of the other.  The performance
of reliable protection tests has been made possible by the production
of sticiently high titer antistreptococcus sera, and by the possibility
of raising the animal virulence of a certain number of strains to a
high degree.  The types of Strepticoccus hamdyticus have been noted
asTypesS3,S23,S60, and S 84, from the serial numbers of the rep-
resentative strains. This nomenclature is not put forth as a final
one, since we realize that probably many other human types exist,
to say nothing of the bovine and cheese varieties, and that the pro-
portional distribution of the different varieties pathogenic for man
may be very different from that represented by this work.  Strep-
tococcus is the largest of all pathogenic groups of bacteria and many
years will be required to bring out the information necessary to the
perfecting of an adequate classification.

  It is of considerable interest that all the members of Type S 60
.  ferment marmite, and that none of the members of the other groups
so far encountered ferments this sugar.  A few unclassi&ble strains,
however, have been found to be mannite fermenters.

This work has cleared up a number of points about Streptococcus
hmdyticus which have been in dispute for many years.  In the first
place, Streptococcus hmwlyticus of human origin is not a unit type as
was previously supposed, but probably consists of a number of types,
at least four of which have been definitely identified.  Previous in-
vestigators have stated that freshly isolated human strains change
their antigenic properties on animal passage, and that the latter pro-
cedure for the development of animal virulence gives a common
antigenic character to all strains. We have found no evidence to
support this contention; in fact immune sera produced with human
strains that have never been passed through animals afford a high
degree of protection against strains that have received many animal
passages.  In addition, the antigenic differences between strains of
Streptococcus ~molyticzcs which have been passed through animals
are as distinct as those between strains which have not been so passed.
The types of Streptococcus hmwlyticus studied have been obtained
almost exclusively from the respiratory tract and from a limited source
of supply, and there is some reason to believe that those which pro-


A.R. DOCEEZ, 0. T. AVERY, AND R. C. LANCJDIELD     213

duce cellulitis, erysipelas, and septicemia may be of somewhat dif-
ferent character.  It is, therefore, readily seen that only a beginning
has been made in the classification of Streptococcus hmwlyticus, and
that before the classification is complete and the relative dominance of
the different pathogenic varieties determined, much work must be
done.

SUMMARY.

1. Immunological ditrerences have been shown to exist between
strains of Streptococcus hamdyticus of the human type.
2. Four biological types have been identified by means of the re-
actions of agglutination and protection.
3. At least two other types have been encountered and the indi-
cations are that more exist.

BIBLIOGRAPHY.

1. Schottmfihr, H., M&u%. nud. IV&., 1903, I, 849.
2. Adrews, F. W., and Horder, T. J., Lancct, 1906, ii, 708. Holman, W. L.,
   J. &fed. Research, 1916, xxxiv, 377.  Blake, F. G., ibid., 1917, xxmri, 99.
3. Smith, T., and Brown, J. H., J. Med. Research, 1914-15, xxxi, 455. Jones,
   F. S., J. Exp. Med., 1918, xxviii, 253. Ayers, S. H., J. Ba~terioL, 1916, i,
   84. Avery, 0. T., and Cullen, G. E., J. Exp. Med., 1919, xxix, 215.
4. Smillie, W. G., J. Ivzject. Dia., 1917, xx, 45.  Cole, R., and Ma&&m, W. G.,
   J. Am. Med. Assn., 1918, lxx, 1146. Levy, R. L., and Alexander, H. L.,
   ibid., 1918, lxx, 1827. Opie, E. L., Freeman, A. W., Blake, F. G., Small,
   J. C., and Rivers, T. M., im., 1919, lxxii, 556.
5. Marmorek, A., Anti. Inst. Pasteur, 1895, ix, 593.  Aronson, H., Deutsch. med.
   Woch., 1903, xxix, 439. Neufeld, F., 2. Eyg. Y. Injectionskrankh., 1,?03,
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