A STUDY OF MANY STRAINS OF
STREETOCOCE!

 

Thesis for the Degree of M. S.
AC [oma oly
ard
F

¢

 
 

A STUDY OF MANY STRAINS OF STREPTOCOCCI WITH
SPECIAL REFERENCE TO THE STREPTOCOCCI
ISOLATED FROM CASES OF BOVINE

MASTITIS.

at a

THESIS

atea

Submitted to the faculty of the Michigan Agricultural
College in partial fulfillment of the requirements

for the degree of Master of Science.

By

?
,

Lloyd R. Jenes.

oto

December 1917.
THES ®
CONTENTS.

Introduction.

Review of Literature.

Method of Investigation. .

Experimental Work.
Morphological and Cultural Characteristics.
Hemolysis.
Vermentation of Saccharine Broths,.
Homologous and Heterologous Agglutination
and Complement-Fixation reactions: upon
Animal Inoculation.

Summary.

Acknowledgement.

References cited.

100213
ol at
 

INTRODUCTION.

Few, if any pathogenic organisms can lay claim to
wider or more multifarious activities than the streptococcus.
The list of human and animal diseases with which the
streptococe? are associated as the main and primary cause
is already a long one and probably not yet complete. In
addition te their conspicuous role as imitators of very
diverse pathological conditions, streptococci are very often
present in mixed or secondary infections. Streptococei occur
in the healthy human subject both on the skin and in those
cavities which open on the surface of the body as the
alimentary canal and nose. They are to be found in the saliva
and feces and have a wide distribution in air, soil and well
water in nearly all pathological conditions of the throat of
whatever nature streptococci are to be found either as the
primary cause or as an associated infective agent. These
organisms are nearly constantly present in secondary infections
in scarlet fever.

the epidemic of streptococcus sore-throat that prevailed
in Chicago during the winter of 1911-12 was definitely traced
to one dairy. Among the herd supplying thés dairy was found a
number of cases of mastitis. Rosenow (1) reports that a type
of streptococcus slightly hemolytic and corresponding to the
pyogenes type, only after artificial cultivation, occurred in
predominating numbers as the etiologic factor of the sore-

throat epidemic. In Boston (2) and in Baltimore (3) likewise,
o20
epidemics have occurred corresponding to the one in Chicago,
milk borne and with similar streptococci.

Davis and Capps (4) of Chicago in experimental work
have caused mastitis in the bovine by the injection of
hemolytic streptococci of human origin. These with a number
of similar observations tend to show relationship in the
etiology of septic sore-throat and bovine mastitis.
Observations would indicate that both conditions are caused
by the same organisnz and it is the purpose of this paper te
present a study of the streptococci collected from both

sources and other available strains.

Review of Literature.

Streptococci were seen in unstained pus by Klebs in
1872. Several years later Koch (5) demonstrated them in
stained sections and in inflammatory exudates. Pasteur (6)
appears to have been the first to cultivate the streptococci
from cases of puerperal fever and to differentiate them from
staphlyocci both morphologically and by the character of the
lesions which they excite. Rosenbach (7) studied the organisa
in great detail and introduced the name Streptococcus pyogenes.

The question of the differentaation of the streptococci
which soon arose after the first description of the organisus
is still unsettled. Numerous researches fer the past twenty
years having failed te present any method or system for proving
or disproving the identity of the many strains of streptocecci.
The large variety of pathological conditions to which the

organism can give origin and its occurence as a saprophyte
 

230

on the healthy human tissues has for a leng time attracted the
attention of bacteriologists. "One and the same strain of
streptococcus may at different stages in its career produce
now a localized suppuration and now no effecta at all," say
Andrewes and Horder,.

Besredka -quesew-that, "it may therefore be accepted as
an established fact that a streptococcal infection may assume
different clinical manifestations depending upon the resistance
of the person infected and the source whence the organism was
originally derived.® |

Considerable discussion has arisen concerning the unity
or plurality of types éncluded within the species known as
Streptococcus pyogenes. iiarmorek (8) and others have steutly
maintained the "Binheit" theory. Considerable evidence in
favor of this view has been advanced by Koch and Petruschky
(9) who showed that a streptococcus obtained from a fatal
puerperal sepsis caused erysipelas in a rabbit when it was
injected subcutaneously, pesdtunests when—fe-was injected
_anbowennecasty, peritonitis when injected intra-periteneally
and septicaemia when injected intravenously. the conclusions
were that the type of lesions produced by Streptococcus
pyogenes depended largely upon the virulence of the culture,
the tissue invaded and the mumber of organisms. Additional
evidence of the "Einheit® ef streptococci has been brought
forward by Resenow (10) who states that he has changed
streptecocci te pneumococci and back again by special methods
of culture and animal inoculation.

The important question for the moment is, Do these
changes of virulence, et cetera, exhibited by the streptococcus
o4<

influence the diagnostic aspect of the question? Theobald
Smith has admirably summed up the present status of the sudject
in the fellowing words: “Spontaneous changes in the cultural
characteristics of the streptococcus do not proceed rapidly
enough, if they go on at all, to interfere with current
bacteriological methods. Tendencies toward slow changes may
be used as further valuable distinguishing characters" (11).

The marked pleomorphism of the streptococcus also led
many observers to classify these organisms according te their
microscopic appearance. Von hi ngelsheim has made the
morphological distinotions paramount and thus proposed the
groups Str. longus and Str. brevis.

The English bacteriologists were the pioneers in invest-
igating the fermentation activities of the streptococci with
a view to ascertaining whether essential differences such as
would be of value in classifying could be observed. Working
upon the assumption that the fermentative powers are bielogical
characters of fundamental importance, Gordon and Andrewes
and Horder employed certain fermentable substances in culture
media. A complete discussion of the grouping according te
action upon carbohydrates and allied subetances prefaces

the "Fermentation reactions" given under experimental work.

Method of investigation.

The cultures from milk samples have been isolated from
plates by picking off colonies of characteristic appearance,
subculturing and subsequently repleting, after microscopic

examination in order to determine purity of the culture.
 

 

 

 

oH<

throat swabs from sore-throat and scarlet fever patients
were immersed in five c.c. of sterile physiological salt
solution. The salt solution blank with inoculum was shaken
and streaks from the suspension made on the surface of human
bdlood-agar plates. After twenty four hours incubation at
37°C. the characteristic colonies were picked off and sub-
cultured. The salt solution blank dilutes the inoculusa to
such an extent that ordinarily colonies are distinct upon the
plate, convenient for removing. The use of the human blood-
agar plate offers two advantages, first, vecause streptococci
grow vigorously upon such a medium and second, it affords an
immediate comparison of relative numbers of hemolyzing and
non-hemolyzing types present in the infection.

In the case of pus samples, loops of the material have
been placed in salt solution blanks and the method followed
as outlined for throat swabs.

Thirty-eight strains have been studied culturally and
twenty two strains employed in the immunological reactions.
The source from which the cultures have been derived is
given herewith:

1. Diseased udder. Chronic mastitis.

Ze do do

Se do do

4. do Acute mastitis.

5. do Cow had had an attack of mastitis

some time previous.
Milk apparently normal.

6. do Chronic mastitis.
7. — do Samples from Grand Rapids, Mich.
9.
10.
ll.
12.
13.
14.
15.
16.
17.
18.
19.
20.
le

256
24.

266
27.

Diseased udder.
do
do
do
do
do
do
do
do

 

ofa

Sample from Grand Rapids, Mich.
Acute mastitis.

do
Chronic mastitis.

do

These four strains were isolated
from samples of "gargety” milk
from Portland, Mich. Mastitis
of an infectious nature assumed
the proportion of an outbreak
among the herd. <A few of the
cases were fatal.

Streptococcus lacticus. Laboratory stock culture.

Streptococcus.
do
do
do
do
do
do
do
do
do

do
do
ado

Sore throat.

do
Sore throat with La Grippe.
Sore throat with Scarlet fever.
Sore throat.

do

do
prom same patient as No. 24.
Sore throat of chronic type.

Normal throat, patient had an
abscess of the gum.

Normal throat.
hemolyticus. Stock culture.

pyogenes. Stock culture,

Streptococcus from sputum.

Streptococcus stock culture,

Streptococcus pyogenes. Pyemic arthritis of bovine,

Streptococcus from endocarditis. Human.

Streptococcus hemolyticus, stock culture.

Streptococcus metachromatos, stock culture.

Streptococcus isolated from contaminated milk.
x
38. Streptococcus isolated from equine peritonitis.
Sequel of castration operation,
organism recovered in pure culture,

During the first part of this study the cultures were
kept growing in dextrose broth containing one per cent of
calcium carbonate which served to prevent the accumulation
of acid. Later bovine bloodeagar was used. The method of
preparation of this medium was as suggested by Bernstein (12).
One hundred cubic centimeters of blood were drawn from the
jugular vein of a cow into a 200 c.c. Erlenmeyer flask
containing 7.5 cece of a one per cent solution of ammonium
 exalate and 125 c.c. of formalin (forty volume strength}.
The blood mixture was shaken for two minutes. The ammonium
oxalate prevents clotting, and thirty minutes are required
for sterilization. The blood was then distributed into
smaller flasks and twice its volume of sterileo.? per cent
Salt solution added. This gives a dilution of 1 to 2400 of
the formalin. The small flasks were sealed and kept in the
ice box until needed. Plain agar 6.6 per cent acié) tubes
were thoroughly melted and then cooled to between 50° and
60°C. and one part of the diluted blood was added to fifteen
parts of the media, which gave an approximate dilution of
2 to 36000 of the formalin which is not strong enough to
interfere with the growth of orgmisms upon the mediun.

After slanting, the tubes were incubated to insure
sterility. Transfers of the stock cultures were made at
intervals averaging seven days.

In undertaking the Immunological reactions, twenty two

strains of quite definite fermentation characteristics were
 

o8<
selected for rabbit inoculation. Pre-injection tests were
made to obtain a record of non-specific reaction both for
agglutination and fixation of complement. Naturally, of
primary importance was the perfection of a technic which would
give uniform results. After the period of immunization blood
samples were taken which were tested against the homolegous
serum and also against the twenty-one heterologous sera.
This work was thought worthy of undertaking from a standpoint
of perfecting a suitable technic and its possible application
in differentiation of strains or in determining the specificity

of immune sera to their respective antigens.

EXPERIMENTAL WORK.
Morphological and Cultural Characteristics.

The streptococcus group comprises those spherical bacteria
in which as multiplication proceeds the successive planes of
division are parallel and the individual cells remain adherent
in longer or shorter chains. The term, streptococcus, there-
fore is purely morphological. Wo help in the classification
of the streptococci is afforded by the form of the individual
cell, since under favorable conditions all appear as regular
spheres. Irregular oval forms occur at times, particularly
in cultures freshly isolated from the throat but the form
usually becomes normal after cultivation. The streptococci
exhibit a range in size from0.5 to 1 micron in diameter with
considerable variation between individual cells in the same
culture. Wo relation has been found to exist between size
and age of the culture or conditions of cultivation,

The organiene remain adherent in chains which vary in

length from four to a hundred or more elements, in which a
<9e

definite association of the cocci in pairs with their
proximate sides flattened is occasionally observed. The
number of elements in the chain varies somewhat according to
the origin of the culture. It has been observed that
streptococci freshly isolated from lesions tend to occur in
longer chains, while those organisms which grow habitually
upon the normal surfaces and muceus membranes of the body
appear more frequently in shorter chains.

Hopkins and Lang (13) in an extended study of many
strains based their records of chain lengths on smears of
the sediment of twenty-four hour dextrose broth cultures. -
Those forming chains of less than ten members were classed
as brevis, those of ten to fifteen members as mediug and those
of more than fifteen were classed as longus. Those strains
forming very long chains covering several fields have been
designated as longior. A similar plan has been used in the
chain length observations in this study. In most smears
voth long and short chains have been found so it is difficult
to utilise this classification but these terms are of value
mainly in a comparative way.

Table I gives the chain lengths for the strains used
in this study. Observations were made from smears of the
sediment of a twenty-four hour dextrose broth culture and
from the water of condensation of a four day blood-agar

culture.
ole
Tabie I.

Mar.: y 3'June Ug « une
Ho, 'Source'Broth prot! Broth’ Broth ‘Broth ‘Bro th" Broth

 

Tpiea ' .L .  L4 L i M ' «M + 5B B
teased ’ t ’ % , t *
’ ?

' ' , ’ %

 

ae eus ’ e t ' 9 9
.' pore M * MY L , Lb ¢°' & v8 YY 8B
t t ' '

 

 

 

 

 

 

 

 

 

 

, at’ ’ ’ ' ' ’ '
eo TY B a BS B . . WM em * a
29.'Hemo- ' M ' Mf L ' M ‘MM Ler Bent B

"lyticus ? ’ , ' ’ ’

Pyogenes ¢ ¢ v SE v , Bp 3

um
2.'Stock ° ' ’ L t« Bteeki » BF B

‘Cult. ° ? ’ > . ’ ’

35. "Pyogenes if a B Y #B ve Faw % B
34.'Endo- mi * «ik lf L , TL * w ft MT UB
"carditis ’ , ‘ ¢ ® ’

356 poeee ’ ’ , iM * MM ¢ ‘ , M

q e t e e e
36. es ' , M ' , ’ 9 B

*shromatos ’ ’ ' , ’ ¢

eee M a x ' «x , 8B , : , B
¢ e ' e 9 Q

peritonitis

 
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It appears from the feregoing table that the record of
chain length does not afford a reliable basis for classification
primarily because cultural conditions greatly modify the
length of chains formed,

Previously, a long chain streptococcus has been
considered as more virulent than one of short chain type.

In this study it is to be noted that saprophytic strains are
essentially of short chain type, bvut not all strains from
active lesions are of the long chain type. Numbers 35 and 38,
isolated from active lesions and of active hemolytic properties
show short chain type in contradistinction to the generally
accepted theory regarding the relationship of long chain
formation and virulence.

There is in general a tendency for shorter chain
formation after a prolonged period of artificial cultivation
which tends to be more marked upon solid media than with
broth cultures, however there are frequent exceptions te that
tendency for in a few instances the reverse difference has
been noted.

Typical streptococci are non-motile, non-flagellated and
do not produce true endospores. Ellis (14) reports the
finding of spores and flagella in streptococei. The
experiments carried on by Bllis would rather indicate the
presence 6f specially resistant cells in old cultures of the
cocci. His figures are by no means conclusive as to the
existence of true spores and, in the ahsence of any observation
of germination of streptococcus spores, they can scarcely be
said to exist. Streptococci may exhibit from time to time
cells which are decidedly larger than their fellows.
-
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w12e

They have been designated by Heuppe as arthrospores. These
arthrospores appear to have no unusual resisting powers, and
they are in no sense to be regarded as true spores. It is
very probable they are involution forms. CyE\@.and A.E.
Winslow (14) found that one of the fundamental differences
vetween the parasitic and the saprophytic cocci was their
vehavior to the Gram stain. The psrasitic torm usually
decolorized. These observations were corroborated by Kligler
(15) who in reviewing the wccacae in the collection at the
Museum of Natural History found that of the saprophytic types
seventy four per cent were Gram negative and ae per cent
were Gram positive and the remaining seventeen per cent were
variable. The cultures used in this study were found te be
Gran positive when smears were made from four day agar
cultures. Ho deviation from this rule was observed. -

Andrewes and Horder (17) consider the characteristic
growth in litmus milk one of the most significant tests and
their differentiation between the Streptococcus pyogenes
group and the gnginosus group is based partly on this
criterion. Their six groups of the streptococci are reported

as giving the following changes in litmus milk:

Equinus * fails to clot milk.
Mitis - acidifies milk without clotting.
Pyegenes - acidifies milk without clotting.
Salivarius - clots milk.

nosu - clots milk.

Fecalis - clots milk,
The only changes which the cocei effect in milk are the

production of acid or alkali, coagulation and decolorizaztion
a

Le
 

~13-
of the litmus. When the organism is most active it uses up
the oxygen and reduces the litmus which is accordingly
decolorized and conversely when activity grows less, oxygen
diffuses from the surface making the litmus pink again.
Coagulation is dependent upon the amount of acid formed. MNHo
casein-digesting enzymes are found among the streptococci and
no gas or odor is produced.

In isolating the strains of streptococei from the udder,
samples have been taken from udders showing certain evidence
of infection. Observations were made as to the relative
prevalence of streptococci as compared with other organisms
and of relative numbers of hemolytic and non-hemolytic types
of the streptococcus. Raturally considerable apprehension
existed in regard to the possibility of strains so isolated,
corresponding in type to the streptococcus lacticus (Kruse)
@ Saprophytic type in many cases responsible for the souring
of milk. Sherman, Bvans and Hastings (16) have suggested
that differentiation is roughly made bdy characteristic
growth on litmus milk. They state that the long chain type
of streptococei usually cumile the milk and may reduce the
litmus after tite curdling, but the color is never completely
reduced. With Streptococcus jacticus cultures, however,
the reduction of the litmus preceeds the curdling and is
complete beneath the sharply defined surface layer.
Streptococcus lacticus also lacks the tendency to form long
chains.

With the fermentation reactions it will be noted that

Streptococcus jactiqus cultures correspond very closely to the
characteristics manifested by the fiyogenes group; with the
ol4-

exception that the lacticus cultures are never hemolytic
while cultures of the pyogenes type are invariably oe es
feature paramount in differentiation.

In Table II are given the results of four days
incubation in litmus milk. In no instance were changes
neted after the fourth day which were to be regarded as
eof bacterial origin.
 

15

 

 

 

 

Test not made.

Table II.
‘ a ~~WWar. ‘Mor. bay'uune'July’
Ne, '. Source vi. '28. ' 3.' 6." 7. °
l. *° Diseased udder * ac ' aC * AC* AC * AC *
2.’ do do * ac * AC * AC* AC * AC !
3. ' do do * ac * AC * AC*t AC * AC
4. * do do * ac * AC * AC* AC * AC ?
5. ° do de * SL * SL * SL*' SL * SL’
6. ° do do * ac * AC * AC’ AC * AC !?
7. ° do de ' SL * 8L * SL* 8L * SL *
8. ° de de "A tA tAYNA * Ac ?
9. ° de de * AC * AC * AC* AC** AC *
10. ° de do * ac * AC * AC’ AC * AC
ll. ° do do * AC * AC * AC* AC * AC *
12. ° de de ‘ac * AC * AC* AC * AC *
13. ° de de * AC * AC * AC@ AC * AC ?
14. ° de de * ac * AC * AC* AC * AC. °
15. ° do do * AC * AC * AC* AC * AC ?
16. ° do do * ac * ac * AC*t AC * AC *
17. ° do do ‘ -« *' A * SL' BL t SL *
18. * Sore throat ' 0 *' C * AC*t AC * AC ?
19. ' do de t .- * AC * ACt AC t Ac *
20, * do de ‘oO tA tA*YA * AC!
21. ° do de "A 'A AYA PA ?
22. ° do do * ac * AC * AC* AC * AC ?
23. ° do do "se *A tAta tga
24. ° do de ‘ee * C *'C* so * AC?
25. ° do de ‘es. *' CC f Cf Alt Ac
26. ° de de *e- YO * O00 * 0 *
27. ° do do 'e *A *'O0'O * QO ?
28. * Normal throat ' AC *' AC * ACt AC ' AC *
29. * Hemolyticus "A tA * A* AC f AC
30. ° FPyogenes "A 'A ' AYA * A!
31. ° Sputum "A *A tTtAPtAaA th *
$2. * Stock culture te to tara ta *
33. * Pyogenes ‘Oo *aAcC* At aA *F A ?
34. * Endocarditis * AC * AC * AC* AC * AC
35. * Hemolyticus , 2s to to * O FAQ ?
36. ° KMetachromatos te Fo * af AC t Ac
37. *° Milk * SL * SL *' SL*' SL * SL !
38. ‘Equine Peritenitis’ A *'‘A tA*A * ACP
Nete: <A = Aeid productien.
C = Coagulation.
© = No change.
SL = Streptococcus 1 us. type of reaction.
-16-

During the period of cultivation as indicated in
Table II, twenty six strains continually gave the same
reaction when plated in litmus milk. The remaining twelve
acquired new characteristics which, for most of them were
the formation of acid and coagulum. A majority of the udder
strains gave regularly an acid coaguilum type of reaction.

Among the sore-throat cultures which, according to
Andrewes and Horder's classification, should clot milk
without preducing acid there were but two that so reacted.
From a standpoint of classification the character of the
growth in litmus milk can scarcely be considered as
satisfactory.

According to Andrewes and Horder's classification
the growth of streptococci on gelatin at 20°C. varies among
the different groups as follows:

equnius -feebdle.

mitis -well,

pyogenes ewelk.

Salivarius -variable.

anginosus -fails.

fecalis well.

Gelatin cultures of the streptococci invariably
present the filiform appearance with very closely aggregated
colonies. No liquefaction was ever ebserved. After several
generations on artificial media there tends to be a more
abundant growth on gelatin. Table III, gives results of stab
inoculations into nutrient gelatin after incubation for four

days at room temperature, approximately 20°C.
eS

 

 

 

Table III.

—nny T ¥ eee F 7 -
No. ° Source. *Mar.'Apr. ‘May’ June' August'
J * 20 *' 20 * 5 ' 20" 15 !'
“t —t —y
1. ' Diseased udder ‘+ ¢' +t et ¢% + '
2.’ de * &#+e Hf» te Pe SF ft (
3. ° do g ¢* + * » t + ? + ?
4. ' do t et +H +t +t + #
5. ° do "+t +h ee ge ?
6. ° do *  t + % + ? + * + ”
7, ° do ' £+- ete fb eee HF He @
8. ° do * @w@»® tt wpt @« €& w& & @ q
9. ' do ‘*+*¢t +74 +t + 8
10. ° do *+4¢t +t et et © ¢
1z. ° do '* +t >» Ff 1 + ¢ ,
12. ' do '— +e &+ ee +e +H He) 8
13. ' do "+t +H Heh ¢h 4 1
14. ° do *“ +e¢ +t eh ~t 4 ’
15. ° do * ¢%* +H et 2 ©
16. * do " ¢t +e ee St @& FC
17. ' Str. lacticus ? ' "¢* +f ¢ t
18. * Sore throat tf © ¢ 41 eh +h +
19. ° do ‘t we fF o2@ % w ? ot e« 9
20. ° do * .« ® wf. tft ot H+ e 0
21. ° do  * -= ht wate th ¢ te 4 t
Zee 0 do 9 oe * a o ¢t + ? + ?
25. ' do ‘oe -* B81’ 81" 81 °
24. ° do * 2. f sp * git +t 4+ ¢
25. ' do * 2» % « * Slt += t 4 t
26. * do t « & @ * 2 tft © & | ?
27. * Normal throat '¢% + Ht ¢H + 8
28. ° do rt © wow FF wo F&F HF ?
29. ' Hemolyticus *.2w.ts tet gt '
30. ' Pyogenes * ¢te (HH HH 4% '
31. * Stock culture ‘* ofrqgeor ¢t ¢ ?
32. * Sputum "* eF wo tet gt 46 ’
33. ° Pyogenes ' +9 @ tet ee 4 ’
34. ° Endocarditis * ¢%*e Het St 4 ?
55. ° Hemolyticus ‘*orogortortr #* ¢ ¢
36. * Metachromatos ‘* ofo froQOFf git + ¢
37. ' Milk "+474 ter gr gs
58. * Equine peritonitis +° + "+" +% ¢ ,

Nete: + = growth.
e = no growth.
81 = slight growth.
All but two of the seventeen udder strains continued
to grow well on gelatin, which would permit of their class-
ifying with the pyogenes type according to Andrewes and

Horder. Of the sore throat strains there was but one that
-18«
grew in gelatin immediately after isolation. Asa
supplementary test, particularly with reference to diff-
erentiation between the pyogenic and anginosus groups,
characteristics of grewth upon gelatin appear to be of very
considerable importance.

Hemolysis.

Hemolytic determinations on blood agar plates
introduced by ‘Schot tauller (39) is perhaps the most widely
used means of differentiating the stveptococei. Originally,
the method of determination consisted in inoculating the
hearts blood of a rabbit dying of streptococcal septicaemia
directly into whole serum and observing the height te which
the ring of dissolved hemoglobin arose.

According te Davis (18) net enough emphasis has
been placed on the property of hemolysis in the general
study of streptococci. Particularly, with strains isolated
from diseased udders as the hemolytic property is an
invaluable means of distinguishing between saprophytic types
and many ef the pathogens.

Numerous workers have attempted the classification
of the streptococci solely upon their characteristic
reactions upon some sort of blood media. “Hemolytic, *
“viridans® and “non-hemolytic” are terms that are generally
accepted as characteristic of certain streptococci. All
observers are agreed upon the existence of the hemolytic
and non-hemolytic types, the former producing that clear
transparent zone peripheral to the colony; but fer the second
or viridans groupcthere are many observations that tend to

invalidate this classification. “The hemelytic power
o19-
possessed by the streptococci is another of the subtle
properties developed in direct response te the bio-chemical
conditions of the host," says Winslow.

It is to be understood that the hemolytic streptococel
do not constitute a single variety of genus. Hemolysis is
a property common to a number of kinds of streptococci that
might differ from one another in a number of other properties.
It is variable at least within certain limits, but nevertheless
sufficiently stable to be a very useful property for many
practical purposes.

Some workers believe the greenish coloration of the
viridans colony te be due to the action of the cocci upon
the muscle sugar present since in sugar-free media the
action may not appear and on glucose bleod agar even the
hemolytic types may show this greenish coloration.
Nevertheless, it is an observed fact that greenish colenies
do result upon the cultivation of certain strains of
organisms, and at the present time this type of organism is
recognized as Streptocoocus viridans though we have not at
present any binding or standard classification.

Methemoglobin is a) intermediate product in the
reduction process of oxyhemoglobin and it is this reduction
that takes place in the formation of the gteenish coloration.
This phenomenon occurs only when the s€reptococei are living
and only in the presence of certain nutritive materials and
is to be explained by the alteration of the exidation
precesses by the organisms in the vicinity of the red blood
cells and not to the production of any injrious substances

capable of isolation. Further, we can also see at once that
-20-
the pathologic effects of bacteria on other tissue cells may
be due to disturgance in the oxidation in the immediate
neighborhood of the cells and not necessarily to the action
of any excreted or secreted poison. It is believed that
this theory may be applicable to the effects caused by the
Streptococcus viridans because the lesions produced by it
are prone to be localized and associated with the presence
of the organisms in the lesion. The evidence is also
supported by an elimination of other factors: As,
methemoglobin is produced in the presence of sugars that
are not fermented and so it is not dependent upon any
intermediate or and products of sugar fermentations; it
is not due to the production of carbon dioxide as this gas
produces in hemoglobin a cherry red pigment; it is not due
to the production of acid as this reaction is somewhat
accelerated wnen acids are removed by potassium phosphate.
The phenomenon of the alteration of the oxidation and
reduction processes wnen stimulated still further results
in an active hemolysis of which the hemolytic group is
capable accoréing to one explanation. And again, the
hemolytic area or clear zone surrounding a colony may be due
to certain proteolytic enzymes which emenate from the colony
and attack the red blood cell, either changing the state
of the membrane or severing the relations of the hemoglobin
from the stroma of the erthrocyte.

In determining hemolysis in the laboratory, the
blood of various species has been used, though only a few

comparative studies have been made. Guinea pig blood has
e2le-

veen reporteé by a number of observers as unsuitable because
of the rapid disintegration of the corpuscle. Kerner (19)
found that the corpuscles of the dog are most easily
hemolyzsed while human and frog are the most resistant.
Becker (20) in a comparative study of sheep, goat, horse,
rabbit and human bloods found that variations were
especially marked in the macroscopic appearance of the
hemolytic zone. Irregular differences were noticed in the
extent of the hemolysis, with the various kinds of blood.
Methemoglovin was formed most quickly on rabbit blood.
Human blood appeared to resist hemolysis by weakly hemolytic
strains better than any of the others.

| In the routine preparation of nutrient blood agar
for maintaining the stock cultures, defibrinated bovine
blood with one per cent ammonium oxalate solution and
sterilised with one tenth per cent of formalin as suggested
by Bernstein and Epstein (12) was used. A series of
plates was poured with this bovine blood agar, so pres-
erved, and was inoculated from the same culture as a
series of the human blood agar plates. <A comparative

record of the results is given in Table IV.
=226

 

Table IV.
"Tt TT oe | '
No. Source. ‘* Human "Bovine ‘* Bovine

blood 'blood * blood
24 hrs, '24 hrs. ' 24 hrs.

 

 

1.* Diseased Udder

32.' Stock culture
33.' Pyogenes

34.* Endocarditis
35.* Hemolyticus
36.' Metachromatos
37.° Milk
38.*Equine peritonitis

y

¢ ’

g ?

t ,

q ' ¢

0 eo t o 9 + e

2.° do ¢ o ¢ a ’ o 0
3.! do ¢ e ¢ e t eo e
4.¢ do ° - t t - ’
5.° do ¢ o ’ = ¢ a %
6.° do ’ - t ' - ’
7.°  ) ¢ - t ¢ = t
8.* do t es q e ¢ @ 0
9.! do ° + ‘ . t + ¢
10.° do q o ? oe e . 9
11.° do , + t 4 ’ + ’
12.° do ' + ' + t + 0
13.° - ao , + t + ‘ + °
14.° do " + t . t ° t
15.° do ' + ' + ’ + 9
16.° do t + ‘ + ’ + ‘
17.*sStr. Lacticus ' - ' ' - '
18.'Sore throat ’ + ' + ’ + °
19.° ado , + ¢ oe % + g
20.° do q + 9 o ’ + ,
21.' do ' ° t ¢ - ’
22.' do ' @ ? o % e ’
23.° do 9 o t o q oe q
24.' do , V t - q o ?
25.° do t o 9 @ ? - ’
26.° do ' o 9 o ¢ o 0
27." Normal throat ' ° t t ° '
28.' doe ¢ = ? o g e q
29.*Hemolyticus ‘ + ¢t + ? + '
50.’ Pyogenem é + ‘ + t + ,
31.° Sputum ' + t e t + y
9 + ? e g o 9

? + % + ? + ¢

% + e + v + ?

9 + ¥ + q + 0

‘ o ' = v @ g

q a % = % eo @

+ 0 + 6 + 9

 

In some instances hemolysis was noted on the bovine blood
agar plates corresponding to the human biood agar plates,
but only after. forty eight hours incubation. The results do
not agree to a sufficient extent to megit the use of bovine

blood.
-23-

Lyall (21) points out that the determination of
hemolysin production on blood agar plates is of necessity in
no degree quantitative sfnce the hemolysis is subject to
eo many variations, that arise from varying concentrations
of the blood present, hemolysis due to acid production .
and inhibition of hemolysis resulting from the presence
of certain carbohydrates. To obviate these difficulties
and make possible a quantitative determination Lyall added
a definite amount of broth culture to one c.c. of a five
per cent suspension of washed sheep cells and incubated in
a water bath for one hour at 37°C. This method, he
considers, when applied to streptococci gives three typus
of reaction. First, those giving canplete solution of the
erythrocytes. Second, characterized by a change in color
of the suspension from a bright red to a dark brown, due
to a transformation of oxyhemoglobin into methemoglobin.
In the third type there is no solution of cells or change
in color.

ihe cultures reported in this study were tested
for hemolysis according to the method of Lyall. One half
cubic centimeter of an eighteen hour calcium carbonate-
dextrose-broth culture was added in each instance to one
cubic centimeter of a five per cent suspension of washed
sheep célis and placed in 37°C, water-bath for one hour.
Thinking that the reaction might be more easily effected
with a more dilute suspension of the cells, a series of
tubes containing a two and one-half per cent suspension
of cells was run simultaneously. Human blood agar plates

were inoculated from the same cultures and incubated at
24-0
37°C. for twenty four hours in order to have an exact
comparison between the two methods. The results of the
three series are given in Table V. Methemoglobin production
appears to be the predominagt type of reaction and without
analogy to the type of reaction on human blood medium.
The results from the Lyall method do not correspond to the

known type of the organisa.

 

 

 

Table V.
——s "7 ' t
Ho.! Source. ‘Lyall Method.'Lyall Method. ‘Human -blood
‘ *5 per cent '2.5 per cnet ‘agar plate.
’ ' Susp. cells.’ Susp. cells.’
1.' Diseased udder * - ' ”

29.° Hemolyticus
30.' Pyogenes

31 e , Sputum

3$2.° Stock culture
53.’ Pyogenes

34.' Endocarditis
35.' Hemolyticus
36.' Metachromatos
37.° Milk

58.'Equine peritonit

Ze ’ do do ’ o ' o ’ o
3.° do do ' - , o ' -
4. ¢ do do ’ = 0 oe g @
5.°* do ado ‘ M ' o ¢ o
6. ’ do do ' @ ' o ' o
7.° do do °* M ¢ hi ~
8. ’ ado do ’ o ' o ’ oe
9. ¢ do do ' M ' hb ' +
10.! do do '* K ¢ K ¢ ~
11.° do do '! M ¢ - ¢ +
12.° do do ° M ' - ' 82
13.' do do °* M ' o ' +
14.' do do '* @ ¢ ° ’ +
15.° do do '°* M ¢ - ' +
16.° do do ' x ' - ° +
17.'Str. Lacticus ’ KM ’ ~ , -
18." Sore throat , + ‘ + ¢ +
19.° do do ° M ' - ' -
20 e ° do do ‘ oe t eo q -
21 e ° do ado ’ @ t oa t os
22.° do do ° x ' M ' °
23.* do do ' M ' MK ¢ o
24,' do do ! M e - ’ 81
25 e ¢ do do ‘ M ’ os g +
26.' do do '! M ' “ ’ +
27.° Normal throat ' + ' « ' +
28.' do do ' M ¢ x ¢ +
t + t + q +
t + 0 + ’ +
t + ® + ’ >
9 + g o q +
t t Q +
g ? q +
' ’ ’ -
' ' ? ~
? t t «
is ' ' +

o meme +
+eest

 
o25<

In order to obtain a comparison of the ability of
the various strains to hemolyze red blood cells and of the
possibility of green pigment production (common to
Schettmuller's group of Streptococcus viridans )upon blood
media the following technic was used as recently suggested
by Becker (20) and which from the experimental evidence at
hand is the most satisfactory indicator of the ability of
organisms to effect the dissolution of erythrocytes.

Standard agar, as used in water analysis, containing
1.5 per cent agar, one per cent peptone and mede one per
cent acid to phenol-phthalein was used. The agar after
melting was cooled to between 50 and 60 C. and one cubic
centimeter of sterile human blood added for each six cubic
centimeters of the agar base, and the whole thoroughly
mixed by gentle rotation. Approximately seven cubic
centimeters were used for each plate. One drop of a twenty
four hour broth culture was placed in five cubic centimeters
of sterile salt solution which served as a diluting agent
for the inoculum giving isolated colonies upon the plate.
Surface streaks were made and results noted after twenty
four hours incubation at 37°C. Isolated colonies only
were used as a basis in detecting hemolysis.

Most workers advise the use of defibrinated human
blood in preparing the plates. The writer has observed
that the possibility of contamination can be minimized by
permitting the blood to flow from the median cephalic vein
of the arm directly into the liquefied agar which has been
cooled. As the tube is immediately poured sufficient time
does not elapse for coagulation to take place. The
hemolytic reactions of the cultures in this study upon human

blood agar are given in Table VI.
o26-=

 

 

 

Table VI.
y v- § 9 9 Ly
Ho. ° Source * April ' May * June ' Jury August
¢ q 5 e e 6 e 5
1. * Diseased udder * a a a
2. °* do do '! - , - * o ° o t -
3. ° do do ' + t .- t « t . ° -
4. ° do do * = % #© t* 2£ ' 2 ~
5. ° do do * . ‘ -« ¢ « ' . ° °
6. ° do do , = , « * o t = ’ o
7. ' do do ? @ ’ @ 0 = ¥ « ? @
8. q do do 9 « e e 9 o ? o q o
9, ° do do '! + ' + % 4 t + ' 8}
10. ° do do e- ' oe % ¢ . ¢ -
ll. ' do do ' + ‘* +¢ t ¢ * + ¢ +
12. ° do do '° + ‘+ + 8 ¢ ‘ s1 ° §1
13. ° do do '° + ‘i + & ¢ ‘ + t +
14. ° do do '° + '*_¢ % 4 , + ¢ °
15. * do do ' + ‘+ t ¢ * 4 ¢ +
16. ' do do ' + t ¢ % 4 ' + t ~
17. * Str. Lacgicus ' 0 ‘ Oo * - t t «
18. * Sore throat ' + ‘' +¢ t 4+ ‘ + ’ +
19. ° do do '° Vv ‘yt . t . ¢ °
20. ' do do ' + t + § + t . ’ o
21. ° do do '! + * sgl" .- ' . t -
22. * do ado , 0 ’ 0 ° o ® o t o
235.6 ' do do ° = 0 e ¢ 2 ¢ oe 9 oe
24. ' do do '! + t + * g1 * §g1 ° 81
25. ' do do ' t -2« ' e@ t ¢ o
26. ° do do ° - t‘ -« ¢* « t . ¢ °
27. * Normal throat '* + ' -« * o t . t ~
28. ° do do , o ¢ o ¢ o 9 oe % o
29. ' Hemolyticus ' + (+ & 4 ' + ' +
30. * Pyogenes ' + +, + % ¢ t + ' +
31. ° Sputum ' + ‘+ + ¢ 4 ' + ¢ 81
32. * Stock culture '° 0 ‘ otf + * + , 81
33. ° Pyogenew * + ‘+ ¢ ¢ * + ’ «
34. ' Endocarditis ¢ + '‘ € % 4+ ‘ ¢ * +
35. * Hemolyticus ’ 0 ‘'* oOo * + * + ¢ +
36. ° Metachromatos ‘' oO ‘ -« ' « t . t :
37. * Wilk , - t ee tf .« ,‘ . ¢ ~
38. ‘Equine peritonitis + ' + * ¢ t + ’ +

iti, a

In Table VI it will be noted that out of twenty one
cultures which on April fifth were recorded as giving
hemolysis on human-blood agar only nine gave the same
reaction on August fifteenth, after a period of blightly
more than four months artificial cultivation. Apparently

the property of hemolysis is not to be considered as a

 
a27~
permanent characteristic of some streptococci. Immediately
the question arises; Can streptococci acquire the property
of hemolysis when subjected to very favorable conditions
as animal passage? During this period of study none have
acquired the property. Further investigation is nezessary

to elucidate this point.

Fermentation of Saccharine Broths.

The application of the carbohydrate-splitting
powers to classification, which proved of such value in
the typhoid, coli, and dysentery group of bacilli, has been
attempted in the case of the streptococci in a desultory way
by many observers but very thoroughly by Gordan, (22)
Houston, Andrewes and Horder (17). Gordon has selected
saccharose, lactose, raffinose, inulin, salicin, coniferin
and mannite taking into consideration also the reactions
in neutral red broth and litmus milk. The reactions with
these media constitute the “Gordon's metabolic tests" for tke
streptococci. Over twelve hundred strains have now been
submitted to Gordon's tests. Andrewes and Horder have
summarized the results obtained and conclude that while in
themselves the chemical tests are too arbitrary to form a
basis for a systematic classification yet taken in
conjunction with other characters “they afford a clue to
the nature of any given streptococcus which is invaluable.”
As a result of these observations they have roughly
classified streptococci into seven groups. "We venture to
believe that some such conception of the streptococci as
we have set forth is preferable to the idea that they are
all of a kind or that they present a hopeless chaos."
e
@28ea
Gerden's Metabelic Tests.

 

 

@
0
e
M wo Sy >
rit oO e e ri 6
Hos B © . 5 k 88
Types Ore oO e @ e 5 e @ Oo a
of §& ti @ eecs SF H © & et Os
Strepte- So oe 5 &§ ao @ 8 a © bt
Pp oy << Oo rH xt oO Ge + GS O f&
cocci. eP S55 0 PH A A A § @ a, 30
4 2 3 @ # & Do & § gd & BH
Oo Dm 4H MW HH DM ODO Ff w& = A,
Equinus. e = + e# #« © ¢ +¢ e& ee Medius -«
Mitds. o + + + so oe ¢ + «0 #¢+ Brevis -«
Pyogenes, « - + + oo © ¢ - -« ¢ Longus +
Salivarius+ + + ¢ + »» « - -« ¢ Brevis -
Anginosus.+ + + + + 2 © os + + #£4xLengus +
Fecalis. *¢+ + + + e © + + + ¢ Brevis «

 

a

This classification has been attacked on several
grounds. Walker (23) asserted that fermentation reactions
were not censtant in any strain, but vary frem time te time.
Buerger (24) found that such a grouping dees net cerrespond
te the type of pathogenicity of the strain. Winslew (25)
and ethers in this country maintained that the quantitative
determination of the acid produc6éd was essential te an
accurate study of fermentations. Hewever after very extensiv
work on saprephytic types they arrive at no very definite
results. Helman (30) in an extensive study of many strains
of streptecocci used Andrewes and Herder's classification
but with many modifications. More than 2400 streptececcus
strains have been adapted to this scheme. His plan of

Classification is herewith given.
Grau-positive cocci in chains, no capsules.

 

 

 

 

 

 

+. . ee HismOLY cLS eee ee ee
! |
- Loctose | + —.. bchose o_.. 7
4
+ | Mannit “| > | Wannit r 1 iw .nnit. 1 7 Macnit |
I+ Salicin ++ Saticin , + Salicin -+ Salicin - + Salicin ctr Salicin -¢ Sa.:icin -4 Salicin -
if
7 4 | 1 | Sf | |
' Yt i | iJ | { is] \
f i 7 of } ! | !
I Pf ; i | { i 4 ! ro |
te ! | tf | | tf | | | |
7 / | I | | |
: | | P| | |
| (| | t | | ] rf do |
if Vt to | | 4 | | A | |
! - I an | | n | !
! - | +H | | i o | | 5 5 | |
| 1H 4 | | Oo | i o oO !
h D | | lo a |
3 3 a a | I { » p | !
QO o as a | | o> | Q bs ef
2 2. o Oo | 0 ty o + |
re) p@ 5 ao oO oO | rt O oO
w SS a0 » > C Q S| q 68 Ha @
zp Pe SP = tor v a © ®@ 0 y
° OM at o ca cc @ > mS C
3 ao 7 ° Oo r a x | ow rt | | @
a © b> 5 o 3 g 8 B+ qs s 5 x
ry 2 oF I 1G & wn fx 4 os Q S Ss 8 a
e ee 6 6 ee : ° e
$4 Sa h4 Sa $y bey by ba ‘ . e@ e e e
2 ee ree BS Ss + eS oS SS .
Qn no a on aan na Na an
-30=

A summary of the literature reveals the fact that
the classification of streptococci according to fermentation
reactions alone has led to the establishment of a bewildering
number of types. Andrewes and Horder group all these types
under six heads to which they hsve given specific names,
They showed a definite redation between their grouping by
cultural tests and the pathogenicity and source of the
organisms,

J. Broadhurst (26) has shown that in the
fermentation of various carbohydrates by streptococci that
a higher degree of acidity is reached using meat infusion
broth than when meat extract broth is used. Accordingly
meat infusion has always been used in prepafing the media
for making the fermentation tests reported in this study.
Table VII shows that a much higher degree of acidity is
reached when two per cent peptone (Witte's) is used than
when only one per cent is used. The more vigorous growth
with two per cent of peptone present probably makes possible |
a greater utilization of energy derived from the splitting
of the carbohydrate.

 

 

Table VII.

ee T™ TInitial

Culture _Peptone' Dextrose’ Lactose 'Mannite'Plain’ Acidity.
9 ? 0 q g

y vy Vv Vv vO Y
No. 30 *' 1% ° 3.8 * 2.3 ' 1.6 ' 0.4% 0.2
No. 21 * 16 #$$'°' #=%2.4 * 2.1 ' 1.0 * 0.3 * 0.2
No. 30 '° 2% , 5.5 , 307 , 3.0 * 0.7 ° 0.2
No. 21 ' 2% °° 3.4 *§ 3.0 * 2.2 * 0.3 ' 0.2

a
e
.
oe
Ld
-
¢
-
- «
-
oe
«
e *

. .
: e
a
®
‘
. e
. ws .
ry . .
. *
a ‘
°
3°
“4 a:
e
e
- - -te -
°
< om ’ - wits .
@
. .
e..- 8
' 6 .
eo - - @
e
e ®
%
0 e
~ of «@ ° . . > & &e

-
é
eo
* ‘
°
8
‘
’
—~ 6 + e@
*
e

ox oe afte

.
.
‘
«
a
.
e
’ ‘
- . _

~~ a wv

+m - twee
The sugar broth tubes were inoculated with one drop
of a twenty-four hour broth culture from a pipet. This
method was quite convenient and gave uniform results.
Titvations were made after twenty-four hours incubation at
37°C. But very little acid is formed after the twenty-four
hour period as is shown in Table VIII below:

Table VIII.

"t * ’ Te

Culture ‘* Carbo- ‘'24 ' 48 ‘ 72

* hydrate.‘hours.' hours.' hours.
q g t

 

a a ae ee ee ee

g

? a  w q e
No. 30. ‘Lactose ' 2.3 2.3 * 2.5
Wo. 30. ‘Dextrose ' 3.8 *' 4.6 °* 5.0
No. 21. ‘Lactose ’ Zed ’ 2.4 t 2.6
No, 21. ‘Dextrose ' 2.4° 2.9 '* 3.7

Therefore for the remainder of the tests, a medium
containing two per cent peptone (Witte's), 0.5 per cent
sodium chloride adjusted to a neutral reaction was used.

To this was added one per cent of the fermentable substances

used, were of the following brands:

Lactose, J. T. Baker Chemical Co.
Salicin, Eimer and Amand.
Rafinose, Kehlbaun.

Mannite, Merck,

Inulin, Kahlbauz.

It was found th at saceharose wes fermented by all
strains hence this carbohydrate has not been used in the
test. Dextrose, Levulose, galactose and dextrin are of no
value in classification as Buerger (24), Winslow and Palmer
(27), and Artz (28) have found that these carbohgdrates
are fermented by all streptococci. On the other hand none

have been found to ferment adonite or dulcite. Tests for
pe
*
- co 8 SS we, . - « . - . ° ¢ mo =. . o- 27 ef @€ = -
e ‘ e <
. ‘ e - . a
* .
° ‘ses e - ® °

! a . ‘ »

» .
es . e « . ‘ | e
e . os oo ~ abo eo = er + .- =o 0 a= oe. --—: . Sem oe aA . . — ere ww . . ——- - -- asa
+ , ° “ .
‘ '
an) t . > oe ‘ : ‘ . é
o t °
. . r : &
>
vf : ‘ 1 .

- ‘
er ‘ , . "
e ‘ ae * . .
. e “ .
e s o .
. .
.
. ‘
t
e . ' t . * :
,
4 o
~
e c ‘
- oo
- . oe :
e € :
. ’ ? + . . -
. ‘ 7.
. .
. s . :
. : ._ o™e .
o = - .
r e
. , . . ' ‘ -
, oy en ¢ iy . e .
” . r . -

: a . . :
: . . :
+ ‘ - . o t :
‘ - . - 7 oe . .
: ’ . . . . . topo
‘ e@ « : - . -
. ~ - . . . Nee,
* . a * » 8 :
- - - ° ~ .
=32<
the reduction of neutral red was not undertaken. As
reported by Buerger (29), and Hppkins and Lang (13) this
test appears to be very indefinite beeause concordant
results are not obtained in repeating the test.

In the titration for the amount of acid produced,
five c.c. of the contents was titrated against H/20 NaOH
using the first pink of phenolphthalein as the end reaction
A correction was made by inoculating a tube of plain broth
and noting the amount of acid formed, during a like period
of incubation. This plan was adopted to avoid the use of
a coOlon-free sugar broth, partly because some streptococci
grow feebly in a colon broth and also to exclude possible
acid formation from non-carbohydrate constituents.

Following the suggestion @f Hopkins and Lang (13)

I have considered as fermenting organisms only those

which have produced over .& per cent of acidity while those
which produced only .8 per cent or less I have considered
as non-fermenting types, the initial acidity having been
deducted from the titre. In Table IX is given a record of
fermentation reactions of the thirty eight strains with
five different test sugars. These reactions have been

noted over a period of five months.
Table Ix

FERMENTATION OF CARBOHYDRATES BY STREPTOCOCCI.

 

Inulin

Mannite

Salicin

Raffinose

Lactose

 

GT qensny

g eunr
g¢ Lon
Og yorEy

ST ysnsny
g eune

¢ son
Of GOAEN

GT ysndny
g eunr
¢ sEK
O2 Wore

ST yengny
9 eune
¢ ARK

Oc YOIBA

ST qengsny
g eunc
¢ SOK

Oc YASH

+ |
+ I
+ |
+ |

= a =p ==
=a ——_ =<: =
—! => -_ >
=. = =2_> =p
=—=: —_ =_> an
—=2- —_ aD =~
= aan = am
=p = Ld =>
ap = —_—> =
== => =? =>
2 =—. =_> =
=> ean —_2 =.
=——_- ap mp =
= an a7 —_
—Z oo an _2
—_ => => =
= == =p a=
— o> —_ —_
=> =n = =
> = = =

HHULHSAHs
Hei dette
HH R+EHI
KHRERTHEHI

+t rt +¢
tert

pe

-

ce
tt *t

i+ i+4t

Pike tr KRttt
Ite torts
PLP eh ent

FEFHEHEL ELE HEE HEeHe He gta
HEEHEHEFHEE EEE EFETHEE LHI
PEt He tee thee eetetisgi
FEPEEEEHEDE EHH EEE HS

i++
i++
i a
i++

oat
@
@
@
«
0
we
w

i=

 

LS Sore thr
@
@
@
@
@
@
e

Sy
@®
co
ro
_
rf

17
19
20
21
22
2

oh
25

orn uy
Nt No oad date
tose

 

  
   
  

~ Ralfinose Baiiocd

 

T ganeiny
g oun?
¢ fen
O02 YUSTUN

T qenIny
Q eune |
¢ uy

Oz WoOsTy

O¢ USIUA |

it
t+
t+
e+

tit
t+
'o+
fod

0 0 — =
OO -— @«

+!
oh
i +t 8
eee

Rit
+iwtt
#or i
eto + f

(ati
it
a2 +4
ood |

StH atetetite
HHH t ete eeteei+se
tE4 TH HBOLL007T
Fe ee Tot Foot

S&itktttortrtoset

Veeeeaee scenes
HHHH+e hee He tatt
Peete treeett
EREHEHEHEEE HEE HEF
;

-

‘

@
MNetachramotos

 

Kndocarditis
35 Heralytiocts

36

32 Stock guiture

29 Heraliticua
4 Pyogenes
3

246 Sore throat
27 Notral *
30 Pyogenes

31 Sputur

37 Milk

38 Peritonitis

28
@o35<
Tadle IX well sets fortR the remarkable constancy

of the fermentation reactions. Evan after five months

of artificial cultivation all but seven strains continue to

give the same reactions. This is in quite direct opposition

to the report of Bergey (29) who says: “Unfortunately,

the streptococci are very easily affected in their

fermentative powers so that it is not an easy matter to

obtain concordant results upon repeating the tests after

the organisms have been grown in arttficial media for some

time.”

Homologous and Heterologous Agglutination and

Complement Fixation Reactions upon Animal Inoculation.

Ever since it has been known that immune sera often
agglutinate the bacteria concerned in their production,
constant efforts have been made by investigators to determine
the value of this reaction in the case of various bacteria
which closely resemble each other. Streptococci have been
included among the bacteria thus studied and certain facts
have been learned regarding the power of the sera to cause
their agglutination, BSignificant antigenic dissimilarities
among closely related streptococci have been determined
by the agglutination test.

The relationship of streptococci to disease as well
as the relationship of different streptococci to one
another was at first based upon the results of the
agglutination reactions. In 1902 Aronson(31) made very
complete observations upon the agglutination action of the
sera of horses which had been immunized. The streptococci

employed in the tests and the immunization were rendered
a36<

highly virulent by multiple passage through mice. He
found that the serum from a horse treated with a certain
streptococcus agglutinated other cultures as well as the
hemelogous one. His observations point to a close relation <
ship between all streptococci. Andrewes and Horder found
egflutination reactions “troublesome and disappointing" but
attempts were soon directed toward a correlation of
agglutination and fermentation reactions. floyd and
Wolbach (32) concluded that fermentation reactions could be
used to separate streptococci into large groups, while
agglutination reactions merely emphasized the individuality
of the members. Kligler (33) om the other hand found that
the agglutination reactions ran strikingly parallal with
fermentation reactions, Though the agglutination reaction
was not found to separate the streptococci into large group
he says: “the agglutination test tends to show that a
division of the streptococci on the basis of hemolysis is
not warranted, whereas, a separation according to the
fermentation reactions appears to coincide more closely with
their natural relationship." Swift and Thro (34) report
that the agglutination test is specific for streptococci but
not specific for individual strains,

Seven years ago Besredka applied the complement-
fixation reaction to the study of horses which had been
immunized to streptecocci and found that the serum of such
horses gave a fixation of complement. The various strains
were specific. Fox and Mallein (35) studied scarlet fever
patients. The sera from ten of the twelve patients with

scarlet fever gave positive reactions, with nine controls,
37
among which were four of erysipelas and one of puerperal
sepsis, which gave negative results.

From the foregoing it will be seen that the question
of classification is unsettled when undertaken from an
immunological standpoint.

Naturally of primary importance in the technic of the
agglutination test was the preparation of a satisfactory
antigen. Attempts to prepare an antigen by growing the
organisms on solid media dnd suosequently washing off with
salt solution were not successful. Growth of these
organisms on most solid media is scanty. All agglutination
antigens reported inthis study were prepared as outlined
by Hiss (36). Erlenmeyer flasks containing 250 c.c. of a
“2 per cent peptone meat infusion broth with 1 per cent of
dextrose and 1 per cent of calcium carbonate were sterilized
by the Tyndall method and subdsequently inoculated. These
flasks were incubated at 37°C. for four days and twice each
day the flasks were shaken. The shaking serves the purpose
of breaking up the longer chains so after the first twenty
four hours the cultures becomes uniformly turbid. At the
end of the incubation period the flasks were shaken and
then set aside for one hour during which time the calcium
carbonate settles to the bottom of the flask. The turbid
broth culture was then pipeted off with a large pipet
attached to an aspirator. The antigen was then diluted
with @6terile salt solution until in density it closely
compared with tube No. 2 of McFarland's nepholometer (37)
and then stored in bottles with .5 per cent of formalin

added as a preservative.
-380
It is a well known fact that formaldehyde even in
very emall smaunts will exert a peculiar action on proteid
material, hardening it or otherwise altering its chemical
and physical properties. In order to determine the amount
of formalin which would interfere with agglutination a
known positive serum was drawn and added to a series of
tubes with antigen containing varieus amounts of formalin.
The formalin was allewed to remain in contact with the
antigen for a period of thirty minutes before plecing in
the tubes for the agglutination test. Inasmuch, as the
action of the formaldehyde might be more pronounced after
a longer exposure, a@tirer-serics—of_tudes—wee-run—etter
a-longes—enreewms, another series of tubes was run after
twenty four hours exposure to the formalin. <A third series
was also run after several days exposure at ice box
temperature. Results of the three series are given in

Table X.
o39-

v Per cent’ Length — — -

, ‘of for- ‘of time °
Antigen'Serum'mealin ‘formalin’

No. ° No. ‘present. 'present.'1:50'1:100'1:200'1:33
. e q q

 

 

   

=

 

v
e ° q e e
5 | 165 0.5 30 mine’ #+°* + * + Y + % 4 , -
9 * 165 * 1.0 * 30 min.' +° + * + * ¢ % + % «@
9 ' 165 * 2.0 * 30 min.e' +°* + * ¢ © +: &© ¢ «
9 * 165 ' 3.0 °* 30 min.' +* + * + * P t «© * f&
9 ' 165 ' 4.0 * 30 min.'§ +* + *&% #€ t 2 % | ¢
9 * 165 * §.0 * 30min.’ +* P & © * @© % 2@ 4%"
9 ' 165 ' 0.6 *¢* 24hre.' +* #¢ * + * # + % «
9 * 165 ' 1.0 * 24 hre.e' +°* + * + * #¢ * P tf «
9 * 165 * 2,0 ' 24 n4e.’ +¢+° + *§ + * Pt ws * «
9 * 165 * 3-0 * 24 hrs.! + ' + °? + ® o ° o ? -
9 ‘165 * 4.0 ' 24 hre.'’ ¢+°* + * + % w@ %* w@ ¢ «
9 * 165 * 5.0 * 24 hrs.*' +* + * PY &@ * & * «
9 * 165 * 0.5 ' 7 days.’ +¢* + * + + + f 2
9 * 165 * 1.0 ' 9? days.' ¢+* + * ¢ %* #¢ | pf «
9 ' 165 ' 2.0 ¢* 7 daye.’ €¢+°*° + * + © + & Pp ft .
9 ' 165 * 3.0 '°* 7 days.' +* # * # * BP tf 2 * f
9 * 165 ' 4.0 ° 7 days.' + + * P &t «= * fs # o
9 ' 165 *' §.0 ¢* 7 days.' +°*° +#¢ * P tt 2s % w& * «

{
|
|

Hote: + Agglutination.
P Partial agglutination.
~ No agglutination.
From the foregoing table it will be .7noted that
l per cent of formalin does not interfere with the
agglutination of an antigen by its specific serum. Twe per
cent of formalin appears to have a slight inhibitory effect
But little difference is noted in subjecting the antigen
to the formalin for one-half Nour or for one week. The
inhibitory effect does not seem to be cumulative.
In running the agglutination test five dilutions
were used. Two cubiccentimeters of the antigen were place
in each of the five tubes and 6.5 cc. of the serum was
added te 4.5 c.c. of salt solution. Frem this 1 to 10
serum mixture 0.4 c.0., Oc2 CeCe, Oel CoCo, 9.04 Cala,
0.02 c.c. were added to tke respective tubes which in the
order listed give approximate dilutions of one to fifty,

one hundred, two hundred, five hundred and one thousand.
-40=
The tubes with control tubes of antigen only were incubated
at 37° C. for at least twelve hours, when results were
read. Complete agglutination is marked by the formation of
clumps or aggregates of bacteris which soon settle te the
bottom of the tube leaving the supernatent liquid clear.
There is however in all tubes a primary deposit of particle
of the calcium carbonate which is not te be mistaken for
agglutination, when comparison is made with the control.

In making the complement fixation test, the simplest
kind of an antigen was used. Fabasks containing 200 c.c.
of dextrose broth were inoculated and incubated for forty-
eight hours at the end of which time the culture was placed
in a large centrifuge tube and centrifuged for fifteen
minutes. The large mass of organisma in the bottom of the
tube was washed three times with phenolized (6.5 per cent)
oat (0.86 per) cent solution. A sufficient amount ef the
solution was added after the last washing te bring the
turbidity of the suspension te compare with tube Ne, 5,0f
MoFarland's nepholometer (37). Antigen se prepared was
feund to have a very satisfactory "antigenic titre" in the
presence of a known immune serum and not undue
anticomplementary titre.

In the routine procegdure of the complement fixation
tests, complement was obtained from guSnea pig's blood.
Blood was taken from the pig's, paracentesis cardji, by
inserting a small hypodermic needle into the left chamber
of the heart. The clear serum was removed and diluted

one to four with physiological ost solution.
e4l-

Varying amounts of the diluted complement were added
to constant amounts of hemolytic emboceptor (1 te 100)
end 0.5 c.c. of a 2.5 per cent suspension of sheep's bleod
cells. In running the test proper, twice the titre or two
units of complement were used as the dose.

Hemolytic ambeceptor was obtained by injecting a
healthy normal rabbit with washed sheep cells as fellows:
April 27, one c.c. of cells, April 29, twe c.c. ef celis
and May 2, two c.c. ef cells were injected intravenously.

On May 4th., or two days after the last injection,
the animal was bled. At that time the serum, diluted
1 te 100 effected complete hemolysis of 0.5 c.c. of a
2.5 per cent suspension of sheep cells in as small amounts
as 0.02 c.c. in presence of complement. This was considered
te be a very atrong serum considering the short period of
immunization and the close proximity of bleeding. On twe
or three different eccasions within the next month 30 c.c.
ef blood was taken from the heart each time and replaced
with physiological salt solution, given intracardially.
Notwithstanding the successive bleedings the serum maintaind
its relatively high titre.

Preparatory to running the preinjection tests for
non-specific reaction of the rabbit sera, the antigens were
each titrated te determine their anticemplementary actien.
Varying doses of antigen were added te two units of
complement and kept in the water bath for 30 minutes at
37°C., subsequent to which two units of hemolytic ambocepte
and 0.5 c.ce of a 2.5 per cent suspension of sheep cells

were added. One half the smallest amount of antigen givginy
o42-
inhibition of hemelygis was taken as the dosage. When
positive serum was available five timem the smallest amount
giving complete inhibitéon in the presence of a positive
serum was used as the dose, Provided of course that the
amount did not interfere with hemolysis in the absence of a
positive serum. |

In making a large number of tests, which was often
necessary, the labor involved in pipeting of the materials
was minimized by adding the preper amount of complement
(1 to 4) te a known amount of salt selution so that the
exact dese of complement was contained in 1.5 c.c. of the
salt solution, which was used in all tubes for dilution.
Likewise, the correct amount of hemolytic amboceptor
(1 to 100) was added to a known ameunt of the sheep cell
2.5 per cent suspension so that 0.5 c.c. of the suspension
contained the proper éose of the amboceptor. The conbining
ef the salt solution and complement and of the hemolytic
amboceptor and sheep cell suspension reduces to a
considerable extent the labor invelved in pipeting.
Titration of the complement dilution made with the hemolytic
emboceptor-sheep cell mixture after being mixed for as long
as eight hours compares with that made before mixing the
materials, so that uniformity is assured in running
successive tests.

Healthy, normal, average-sized rabbits were selected
for the immunization. Pre-injection tests, beth for
agglutinins and complement binding substances were made.
Suspensions of erganisms for inoculation were prepared by
centrifuging one twenty-feurehour dextrose breth culture

of each organism, remeving supernatent broth and suspending
 

PN
oA3u
each bacterial mass in five c.c. of sterile salt solution.
Four inoculations were given intraperitoneally as
outlined below:
June 16, 3 c.c. killed by heating to 60°C. for
¢ 00 minutes. |
June 23, 5 cece killed by heating te 60°C. for
30 minutes.
June 30, 2 c.c. live suspension.
July 7, 5 cece live suspension.
On gily 16th, or nine days after the last injection
the animals were bled and serum tests immediately made,
The following tables give a record of the serum

tests:
‘+++ !

b
_

 

 

 

 

 

_a—-/
_.
a _
— =
a =
-
w -¢
—_ a
—_ -
~~ =
-_ -
—
—_— =_
—-
= ea
a =
~— ae
- —_
- —_
a =
= =

-

ate

oe

>

Va «*

-r- ms
o--_
- oa
ao =
- =

- ~
- -

Sd
-—-

- o-

e =~
= -_
en ew
—- of
—_ &
_w
=a
es
e -e - - .
'
' .
' . .
, 2 e
* ' . -
a *
.
« s a e a e @ a a »
. - - - . 2@
. . ‘
-_- -
. e ‘
~~ = = _—_— =
= , «
.
— -

’ . . . a
. -
—_ ep —_— -_- .
° . . '
. ® * . -
e 1 -
- «+. ~—~ - ,
, a . . .
.
- -
. . ° ®
— om - - -—~ _—_— &»
. * °
- = —_ es. —_— «@ - — a7 ~ ~
: . .
_- =—~ -~— >a > . -
° ‘ e
~— . - «: - - = an “- —_— «=
: . @ .
— ~ . wee _ ° - - o
2 ¢ . > ‘
—_— 2m - . — ’ ~
.
® . ¢ q ’ é
me ‘ an - - - - -
y - . ° ‘ :
- - . =e _ -_ _ a
. * ’
. ~~ =! = ~ = - = ms iw
“
~— - am +. —_J e- —_ - -_— -_
\ 4 . .
a - . m= - - we
* ° ‘ ° <
=~ = -_ - - . -_ « - e =
. ’ .
- . ~ _ —- = —_ -
« . . . ~
~45-<
Table XI shews but little strain specificity er ef
groups specificity. From a standpoint of discovering a
satisfactory technic the results are very encouraging but
after little or no hepe of showing the specificity of an
antistreptococcus serum to its own entigen. These results
are much in accord withthe reports of Brown and Hitchens
‘who, after an extensive study of the antigenic preperties
of various streptococci, say in part: “It was very
surprising to note how some immune sera gave cress-fixation
with a large uumber of antigens. Outs results did not seem
to show that the complement-fixation method was of much

service in classification. *

Summary.

From the diseased udders of cows, hemolysing and
non-hemolyzing streptococci have been iselated. These
organisms through their fermentative activities are te be
classified as pyogenes ané mitis. None are to be considered
as belonging to the anginosus group which is considered as
the etielogic factor of sore-throat.

Future research is necessary to demonstrate the
correlation between exaltation in virulence, as by animal
passage, and that of fermentative preperties together with
hemolytic determinations. In the mind of the writer this
should form the basis for very valuable research in determining
the nature of that elusive organism, the streptecoccus.

Agglutination and complement-fixation reactions
failed to demonstrate either strain or group specificity.
In concluding I wish to acknowledge
my indebtedness te Professor L. H. Coeledge
and to Dr. Ward Giltner for mguggestions and

assistance received during this investigation.
o47 =
References Cited.

(1) Roseneu; B. Cc.
1912. A Study of Streptececci from Milk and from
Epidemic Sore Throat and the Effect of
Milk on Streptecocci. Jour. Inf. Dis.
Vol. XI, pe 338.

(2) Winslow, C. EB. A.
1912. An Outbreak of Tonsilitis or Septic Sore
Throat in Eastern Massachusetts and
its Relation to an Infected Milk Supply.
Jour. Inf. Dis Vel. X, pe 73.

(3) Hemburger.
1912. Jeur. Amer. Med. Assoc., Vol. 58, p. 1109.

(4) Davis, D. J. and Copps, J. A.
1914. Experimental Bovine Mastitis Produced with
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Jour. Inf. Dis., Vol. XV, pe 135.

(5)
“678. Untersuchungen uber Wundinfektion.

(6)
1880. Compt. read. Acad. Sci., XC, pe 1035.

(7) Rosenbach. .
1884. Mikroerganismen bei Wundinfektious - Kraukh.
ées Menschen Wiesbdasien.

(8) |
1902. Berl. Klin. Wocheuschr., SXXIX, Pe 299.

(9)

 

~ 2bid, pe 478.

(10) Rosenou, B. C.
(See Kendall)

(11) Smith and Brown.
1914. <A Study of Streptococci Iselated from Certain
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Occuring in Messachusetts in 1913 and
1913. Jour. Med. Res. Vol. 31, pe 455.

(12) Bernstein, BE. P. and Epstein, A.A.
1906. <A Simple Method of Sterilizing Blood for
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p. 772.

(13) Hopkins, J. G. and Lang, Arvilla.
1914. Classification of Pathogenic Streptococci
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Dis. Vel. XV, pe 63.
bd
(14)

(15)

(16)

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| w48=
Winslow, Ce B. Ae .
1904. The Sgatematic Relationship of the
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Kligler, lL. J.

1913. <A Note on the Behavior of the Saprophytic
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Jour. Exp. Med. Vol. XVII, pe. 653.
Evens, A. C. Hastings, BE. G. and Hart, E. B.
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1906, A Study of the Streptococci Pathogenic for
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Davis, D. J.
1914. The Growth and Viability of Streptococci
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Kerner.
1905. Central f. Bacterial I, p. 38, 225.
Beeker, W. C.
1916. The Necessity of a Standard Blood-Agar
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Vol. XIX, De 754.
Lyall, H. W.
1914. Observations on Hemolysin Production by
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1905. Lancet LXXXiii,, pe 1400.
Walker.
1910-11. Proc. Royal Med. and Clin. Soc., Lendon,
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1907, The Differentiation of Streptococai by
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Winslow, C. E. A.

1912. The Classification of the Streptococci by
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@o49e

Breadhurst, Jean.
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Winslow and Palmer.
1910.
Jour. Inf. Dis., Vol. 8, pe l.
Artz. "
Central fur Bakteriologie, Vol. LIV,
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Differentiation of Cultures of Streptococcus
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Holman, We. Le

1916. The Classification ef Streptococci.
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Brown, Claude P,
1917. Extract from personal letter.
May 21, 1917.
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(39) Schottmuller.
1903. Munchener medizinische Wochenschrift
Ho. 20, 21. Die Artuntersheidung d.f.d.
Menschen pathogenen Strept. durch
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