THE DIFFERENTIATION OF HEMOLYTIC STREPTO-COCCI FROM VARIOUS SOURCES BY THE GROUP PRECIPITIN REACTION AND BY BIOCHEMICAL TESTS
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THE DIFFERENTIATION OF HEMOLYTIC STREPTO-
COCCI FROM VARIOUS SOURCES BY THE GROUP
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PRECIPITIN REACTION AND BY BIOCHEMICAL
TESTS
J. B. GUNNISON, M. P. LUXEN, J. R. CUMMINGS AND M. S. MARSHALL
Department of Bacteriology, University of California Medical School,
San Francisco, California
Received for publication October 31, 1939
This study was undertaken, first, to determine the prevalence
of the different serologic groups of hemolytic streptococci in
specimens collected in this vicinity; and, second, to obtain addi-
tional data on the correlation between certain biochemical tests
and the group precipitin reaction of Lancefield (1933). A total
of 561 cultures was studied of which 188 were isolated from mate-
rial from human infections, 53 from normal persons, 8 from
infected animals, and 312 from raw milk.
Several such studies have been made since the Lancefield
technic has been available. A similar survey of cultures from
human and animal sources was reported by Coffey (1938).
Butler (1938), Plummer (1935), Reid and Browne (1939) and
Kobayashi (1939) have studied cultures derived chiefly from a
variety of human sources. Among others who have classified
hemolytic streptococci by the precipitin test are: Hare (1935),
Davis and Guzdar (1936), Foote, Welch, West and Borman
(1936), and Kodama, Ozaki, Nishiyama and Chiku (1938), who
studied cultures from the nose and throat; Hare and Maxted
(1935) and Smith and Sherman (1938), who examined human
feces; Lancefield and Hare (1935), Rolfs, Trussell and Plass
(1938), and Congdon (1935), who collected strains from the
vagina and respiratory tract of parturient women; Colebrook,
Maxted and Johns (1935), whose cultures were secured from
human skin; and Plastridge and Hartsell (1937), Valentine (1938),
689600 GCNNISON, LUXEN, CUMMINGS AND MARSHALL
and Sherman and Niven (1938), who used cultures derived from
milk. Stableforth (1937), Brown (1939), Stewart (1937) and
Lancefield (1934), have studied Group B strains intensively;
Evans and Verder (1938) and Edwards (1934) have differentiated
the human and animal strains of Group C; Sherman (1938) has
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studied Group D organisms; and Bliss (1937) has investigated
cultures of Groups F and G. In addition to the serologic tests
the investigators mentioned and others have employed a variety
of biochemical and physiologic tests. The results obtained
have been summarized by Sherman (1937) in a recent review.
The biochemical reactions chosen for this study included cer-
tain common tests of known differential value and also some
tests, including the fermentation of glycerol and raffinose and the
hydrolysis of starch, which have not been widely used since the
Lancefield method has been applied. Sherman (1937, 1938)
has urged that some of the older biochemical tests which have
been more or less abandoned should be studied again in relation
to the serologic grouping. These tests were carried out with a
representative sample of the Group A strains and with most of
the cultures of other groups.
SOURCE OF CULTURES
The streptococci in this collection were obtained between
1935 and 1939 on the Pacific Coast, most of them from the vi-
cinity of San Francisco. No studies of these strains had been
published previously.
The cultures from human sources were received from Dr. A.
Haim, Miss C. Kohl, Miss J. Stickel, Dr. E. L. Herron, Miss B.
U. Eddie, Dr. C. W. Bonynge and Miss L. Veazie, whose coopera-
tion is greatly appreciated. Most of the cultures from human
infections came from three hospitals in this city. They included
nearly all of the hemolytic streptococci recovered from speci-
mens routinely submitted to the laboratories of two of these
institutions for a period of five months and of the third for about
a year and a half. In addition, the third laboratory collected
most of the hemolytic streptococci isolated which did not fall
in serologic Group A for a further period of a year. In order toDIFFERENTIATION OF HEMOLYTIC STREPTOCOCCI 691
obtain as great a diversity of strains as possible only one culture
was studied from each person. These unselected cultures,
representative of those encountered in routine tests with material
from human infections, should give an approximate index of
prevalent groups.
Cultures were also secured from normal noses and throats of
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staff members of these institutions and of dairy workers.
The strains derived from milk were isolated in this laboratory
chiefly from samples of raw milk shipped to the county for
pasteurization. The results of this milk survey, made with the
kind cooperation of Dr. J. C. Geiger of the San Francisco Depart-
ment of Public Health, will be reported in detail in another paper.
With a few exceptions not more than two or three cultures were
studied from a given sample.
METHODS
The identification of strains as pure cultures of hemolytic
streptococci was made by preparing pour plates and surface
plates in beef-heart infusion agar containing 5 per cent sheep
blood. Serologic tests were made as soon as the cultures were
obtained but some biochemical tests were done later in order to
provide uniform conditions.
Stock cultures were maintained in beef-heart infusion broth
containing finely ground particles of beef heart to a depth of
about 2 inches. After incubation, cultures were layered with
about one-half an inch of mineral oil and stored in the icebox for
as long as eight months. Duplicate cultures were kept by
drying the organisms while in the frozen state according to the
method of Swift (1921).
Preparation of antiserums
Cultures for preparing specific precipitating antiserums for
Groups A to H were obtained from Dr. R. C. Lancefield who also
kindly furnished a supply of antiserum for preliminary tests.
Rabbits were injected according to the method outlined by
Lancefield (1933, 1938) using formalin-killed cultures throughout.
Satisfactory antiserums were obtained after 10 to 30 injections692 GUNNISON, LUXEN, CUMMINGS AND MARSHALL
for all groups except D. After a rest period of six months a
series of 10 to 20 more injections yielded potent precipitating
antiserums for every group. Merthiolate in a final concentra-
tion of 1:5000 was added to antiserums.
Preparations of precipitinogens
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Antigens for macroscopic precipitin tests were prepared by a
slight modification of Lancefield's method (1933). Cultures
were grown at 37TC. for 24 hours in 50 ml. amounts of beef-heart
infusion broth contained in centrifuge tubes and then were
centrifugalized. The sediment was resuspended in 3 ml. of
twentieth-normal hydrochloric acid in 0.85 per cent sodium
chloride solution, containing two drops of meta-cresol-purple
indicator. This was boiled for 10 minutes, cooled, neutralized
with two per cent hydroxide until the indicator turned faintly
purple and centrifugalized. The clear supernatant fluid was
used for the tests.
Certain antigens of Groups C and G which gave marked cross-
reactions were purified somewhat by removing protein material
by precipitation with alcohol or with acetic acid. Three volumes
of 95 per cent ethyl alcohol were added to the extract and, after
standing overnight in the ice chest, the precipitated material
was removed by centrifugalization; the supernatant fluid was
evaporated and the resulting sediment was redissolved in an
amount of 0.85 per cent salt solution equal to the original volume.
As an alternative method, 10 per cent acetic acid was added drop
by drop until no more precipitate was brought down; after
standing overnight in the ice chest, the material was centrifu-
galized and the supernatant fluid was neutralized and used for
the tests.
Antigens for the microscopic method of Brown (1938) were
prepared according to his directions. The cultures were grown
in 5 ml. of beef-heart infusion broth containing 1 per cent glucose
for 24 hours at 370C., centrifugalized if necessary, and all but
about 1 ml. of the supernatant fluid discarded. Two drops of
meta-cresol-purple indicator were added and 2 per cent hydro-
chloric acid was added drop by drop until the indicator turnedDIFFERENTIATION OF HEMOLYTIC STREPTOCOCCI 693
slightly pink at about pH 3.0. The suspension was then boiled
for 15 minutes, cooled and neutralized with two per cent sodium
hydroxide until the indicator turned slightly purple. After
centrifugalization the clear supernatant fluid was ready for use.
Precipitin tests
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In macroscopic tests only one dilution of antigen was used for
each antiserum. To 0.1 ml. of antigen in a small tube 0.3 ml. of
saline was added and 0.1 ml. of antiserum was allowed to run
down the side of the tube gently so as to form a ring. Tubes
were placed in a water bath at 370C. for one-half hour, observed,
shaken, replaced at 370C. for an hour and a half and the results
were recorded. Tubes were then left in the icebox overnight
and the observations were checked. If 0.1 ml. of antigen failed
to react, tubes containing 0.4 ml. and 0.025 ml. of antigen were
used. In order to save material and time many of the antigens
were tested first only with two or more antiserums, suggested by
the source of the cultures as representing the most probable
groups.
For the microscopic method of Brown, the inner surface of the
lid of a Petri dish was ruled in 12 mm. squares with a diamond
point. The antiserum was centrifugalized before use to free it
from all particles. A 2 mm. platinum loop was used to place a
loopful of antigen and a loopful of serum in the ruled areas on
the inner side of the dish and to mix them gently. A piece of
moist filter paper was placed in the bottom of the dish and the
lid placed over it. The dish was placed on the stage of a micro-
scope with the lid nearest the objective and observed with a 16
mm. objective.
Biochemical tests
Fermentation of carbohydrates. The medium used was Difco
tryptose broth base containing phenol red to which 0.2 per cent
agar was added to make it semi-solid. The carbohydrates tested
were sorbitol, trehalose, glycerol, salicin, lactose, raffinose and
mannitol. These substances were made up in 10 to 20 per cent
solutions, sterilized by filtration through Seitz filters and added694 GUNNISON, LUXEN, CUMMINGS AND MARSHALL
to the base aseptically so as to give a concentration of 1 per cent.
The medium was distributed in small tubes in 2 ml. amounts.
Inoculations were made from cultures in beef heart infusion
broth containing particles of meat and the tests were placed at
370C. and observed daily for one week.
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Action on milk. Freshly prepared sterile skimmed milk con-
taining brom-cresol-purple was inoculated, incubated at 370C.
and observed daily for one week.
Hydrolysis of starch. Pour plates were made in beef-heart
infusion agar containing 2 per cent soluble starch. After incu-
bation for 72 hours the plates were tested for hydrolysis by flood-
ing them with iodine solution as described by Andrewes (1930).
Hydrolysis of sodium hippurate. The medium described by
Coffey and Foley (1937) was inoculated with 0.2 ml. of culture,
incubated at 370C. for 72 hours and tested for hydrolysis with
acidified ferric chloride.
Soluble hemolysin. Cultures were grown for 12 to 18 hours in
beef heart infusion broth containing particles of meat. Then
0.5 ml. of culture was mixed with 0.5 ml. of a 5 per cent suspen-
sion of rabbit cells. Observations were made after one hour's
incubation at 370C.
Double zones of hemolysis. Pour plates were made from cul-
tures diluted so as to give not more than 100 colonies in beef-
heart infusion agar containing 1 per cent Difco neopeptone and
mixed with 5 per cent rabbit blood. Plates were incubated at
370C. for 48 hours and were then placed in the icebox overnight
as recommended by Brown (1939).
RESULTS
Of the cultures associated with infections in human beings, the
majority belonged to Group A. Of 188 such strains only 34
fell in other groups. These were distributed among Groups
B, C, D, F and G (table 1). Twelve cultures failed to react with
any antiserum.
In the apparently normal nose and throat streptococci of
Groups A, B, C, G and H were found. A large proportion of
cultures (13 of 50) from the normal respiratory tract could notDIFFERENTIATION OF REMOLYTIC STREPTOCOCCI 695
be identified by the precipitin test, whereas only 12 of 188 strains
from infections remained thus unidentified.
TABLE 1
Classification of Hemolytic Streptococci by the Group Specific Precipitin Reaction
NUMBER BELONGING TO GROUP
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SOURCE OF CULTURES TED -
A B C D E F G H ?
Human-Infections*
Purulent exudates from:
Wounds and skin ......... 19 14 2 0 0 0 0 3 0 0
Mastoid and ear .......... 35 33 0 1 0 0 0 1 0 0
Pelvis .................. 7 4 1 0 1 0 0 00 1
Abscesses-miscellaneous.. 30 25 0 0 0 0 0 3 0 2
Sputum and pleural fluid. .. 8 8 0 0 0 0 0 0 0 0
Spinal fluid ................. 2 2 0 0 0 0 0 0 0 0
Blood .................... 12 11 0 0 0 0 0 00 1
Urine .................... 16 1 5 2 2 0 0 1 0 5
Nose and throat ............ 50 37 3 0 0 0 0 7 0 3
Unknown ................... 9 7 0 1 0 0 1 00 0
Total-Human infections. 188 142 11 4 3 0 1 15 0 12
Human-Normal
Nose and throat ............ 50 14 2 5 0 0 0 13 3 13
Feces ..................... 3 0 0 0 3 0 0 0 0 0
Milk ...................... 312 8 263 12 2 4 0 9 1 13
Animal infections ............. 8 4 0 3 0 0 0 1 0 0
Total-All cultures ....... .561 168 276 24 8 4 1 38 4 38
* The clinical conditions of the persons from whom hemolytic streptococci were
isolated included abscesses of various parts of the body, cellulitis, cystitis,
erysipelas, influenza, mastoiditis, metritis, meningitis, nephritis, osteomyelitis,
otitis media, pneumonia, puerperal sepsis, pyelitis, scarlet fever, septicemia,
sinusitis, tonsillitis and wound infections.
In raw milk the most commonly found hemolytic streptococci
were of course members of Group B, but a few cultures of every
other group except F were isolated.
The macroscopic precipitin test was highly group-specific in
most instances although some cross reactions occurred. More
than half of the Group A cultures gave slight reactions with696 GUNNISON, LUXEN, CUMMINGS AND MARSHALL
antiserums of Group C or G or both. Such precipitations were
much weaker than those with Group A serum and the precipitate
was formed slowly and was granular rather than flocculent as it
was with the specific serum. Precipitation with the specific
serum usually occurred within two hours but the non-specific
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reactions seldom appeared until the tests had stood overnight.
These cross reactions did not seem to interfere with the proper
grouping of the cultures.
About two-thirds of the ordinary antigens of Group C and G
cultures reacted to an equal extent with serums of either group
so that it was impossible to assign them to one group or the other.
However, after the greater part of the protein material had been
removed from extracts of these cultures by treatment with
acetic acid or with alcohol most of them reacted specifically.
All of the antiserums except those of Group H reacted equally
well by the Lancefield and Brown methods. Group H serums
failed to give satisfactory microscopic tests. Many of the rabbit
serums, especially among those injected with Group A or B
antigens, gave strongly positive Brown tests visible to the un-
aided eye within a few minutes. Cross-reactions among Groups
A, C and G were less apparent with the Brown technic and were
never observed when the tests were read within fifteen minutes
after mixing. However, an appreciable number of false positives
which did not agree with the results of the Lancefield tests was
obtained by the microscopic method. The ring test readings
in the Lancefield method were found to be of little value because
non-specific rings sometimes formed which later disappeared.
In the hope that the inconvenience of preparing separate
antigens for the two precipitin methods might be avoided, the
extracts prepared according to the Lancefield technic were used
for the Brown tests and vice versa. It was found that micro-
scopic tests could be satisfactorily performed with the Lancefield
antigen but that the Brown antigen was not suitable for the
macroscopic method.
The fermentation reactions of the Group A cultures were in
general agreement with those commonly reported for this group
(table 2). All but six of the Streptococcus pyogenes strains fer-~ 697
DIFFERENTIATION OF HEMOLYTIC BTREPTOCOCCI
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mented trehalose, salicin and lactose and failed to produce acid
from sorbitol, raffinose, glycerol and mannitol. Three cultures
isolated from mastoid and tonsil infections produced acid from
mannitol in addition to the sugars usually fermented. Three
cultures isolated from a single sample of milk failed to produce
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acid from any of the carbohydrates tested.
The Group B cultures could be placed in three divisions on
the basis of their reactions in salicin and lactose as reported by
Brown (1939). All Group B strains isolated from milk fermented
lactose but more than half of them failed to ferment salicin.
The Group B cultures obtained from human sources all fer-
mented salicin with the exception of one culture isolated from
urine, but over half of them did not act on lactose. A few strains
from milk did not attack trehalose and several of them produced
acid in glycerol. None of the Group B cultures fermented
sorbitol, raffinose or mannitol.
The fermentation of sorbitol and trehalose divided the Group
C streptococci into two classes. Those associated with animal
infections and some of those from milk acted on sorbitol but not
on trehalose, whereas those from human infections and the
remainder of those from milk attacked trehalose but not sor-
bitol. Group C streptococci of the human type varied in their
action on salicin and lactose. Glycerol, raffinose and mannitol
were not fermented by any members of this group.
The few Group D cultures studied fell into two divisions.
Those from the human body fermented all the carbohydrates
tested except raffinose, but those from milk fermented lactose
only.
Group E streptococci produced acid from sorbitol, trehalose,
salicin, lactose and mannitol. The single Group F strain re-
covered fermented trehalose, salicin, and lactose.
Members of Group G varied in their fermentation tests. All
those isolated from milk acted on lactose only. The human
strains which formed minute colonies fermented raffinose in
addition to trehalose, salicin and lactose. The remaining
cultures of human origin all fermented salicin but their action
on trehalose and lactose was variable. Sorbitol, glycerol and
mannitol were not broken down by any of the Group G cultures.DIFFERENTIATION OF HEMOLYTIC STREPTOCOCCI 699
Three Group H streptococci from the human throat fer-
mented salicin, lactose, and raffinose and one of them also
attacked trehalose. A Group H strain from milk fermented
lactose only.
To summarize, sorbitol was fermented only by Group C strep-
tococci of animal origin, Group E cultures and Group D strains
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from human sources. Trehalose was fermented by all the cul-
tures except those of the animal Group C and a few members of
Groups A, B, D, G and H from milk. Salicin was fermented
by every culture of human origin except one Group B strain,
but among the cultures of animal origin over half the Group B
organisms and a few cultures of Groups A, C, D, G and H failed
to react. Lactose was attacked by all cultures with the excep-
tion of occasional strains of Groups B, C and G of human origin
and of three atypical Group A strains from milk. Raffinose was
fermented only by a few cultures of Groups G and H. Weak and
irregular fermentation of glycerol occurred with a few Group B
cultures; Group D strains from human excreta attacked it
strongly. Mannitol was acted upon only by the human Group
D cultures, by Group E, and by three Group A strains. It is
noteworthy that all of the cultures from human sources except
one Group B strain fermented salicin regardless of group and
that all of those from milk fermented lactose with the exception
of three atypical members of Group A.
Sterile milk was curdled by all but four of the Group B cultures
isolated from milk, by all members of Groups D and H, and by a
few cultures of Groups C and G from human sources.
Sodium hippurate was hydrolyzed by every Group B culture,
by the Group D cultures originating from milk, and by the three
atypical Group A strains from milk. All other streptococci
tested failed to attack this substance.
Soluble hemolysin production could be demonstrated for all
cultures except those of Groups D and H by the method used.
The hemolysis was most marked with Groups A, C and G.
Hydrolysis of starch occurred with nearly half the Group A
cultures tested and with all the Group C strains of animal origin.
Starch was not broken down by members of other groups.
Formation of double zones of hemolysis took place only among700 GUNNISON, LUXEN, CUMMINGS AND MARSHALL
Group B streptococci, approximately half of which showed
this phenomenon. It occurred much more frequently with the
salicin-fermenting strains than with those which did not fer-
ment this sugar.
DISCUSSION
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The preponderance of hemolytic streptococci from human
infections fell in Group A and could be identified as Streptococcus
pyogenes, as was to be expected. It seems noteworthy that
from infected persons only 46 cultures which did not belong to
Group A were encountered in the laboratories of two large
hospitals during five months and in a third institution during
two and a half years. The actual proportion of strains of Group
A streptococci was even higher than is apparent from Table 1
because during the last year at the third institution only cultures
not belonging to this group were collected.
Eleven Group B cultures were obtained from diseased persons
but inasmuch as nine of these strains were derived from the
genito-urinary tract or from the throat their presence could
not be considered significant because they are found in these
sites in normal persons, as shown by Brown (1939) and others.
Two Group B cultures present in skin infections might have been
secondary invaders.
Organisms of Groups C and G have been reported rather
commonly in infections of man, but of nineteen such cultures in
this collection not more than ten could be considered to bear an
etiologic relationship to the disease processes concerned.
Group D streptococci were isolated in three instances from
genito-urinary infections where they may have a r6le similar to
that of opportunists such as Escherichia coli. The failure to
find more than one strain of Group F perhaps might be attributed
to the fact that the minute colonies of these orgamems were
overlooked in routine work. Some of the unidentified cultures
might belong to Group K for which no antiserum was available.
The number of cultures which gave cross-precipitation with
antiserums of other groups is somewhat higher than that re-
ported by other authors. In Lancefield's original series (1933)DIFFERENTIATION OF HEMOLYTIC STREPTOCOCCI 701
only five of 106 cultures gave weak cross reactions, Plummer
(1935) reported no non-specific tests among her collection and
Hare (1935) recorded that only four of 100 of his Groups A, C
and G strains gave cross reactions. However, Davis and Guzdar
(1936) noted that ten of 78 cultures gave reactions with both
C and G serums, Kodama, et al. (1938) stated that nine of 58
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strains reacted with both C and G and Lancefield (1938) reported
that cross reactions with Group C antiserum were fairly common.
Butler (1938) found that sixteen of 80 Group A streptococci
reacted strongly with C antiserum and in some instances with
G, but that after purification of the extracts by Fuller's form-
amide method (1938) they precipitated only with the specific
serum. She reported that those strains which gave cross reac-
tions all formed atypical dome shaped colonies and were derived
from mild infections. In the present series at least half of the
Group A cultures showed slight cross reactions with serums of
Groups C or G or both but there was no evidence that such
strains were atypical or that they were associated with less severe
infections than those which gave precipitation with the specific
serum only. It seems evident that the human varieties of
Groups C and G are very closely related antigenically and that
they bear some relationship to Group A as well. However,
the cross reactions were of such a nature that usually they did not
interfere with the practical application of the test.
Although the series was small the comparison of the results of
nasal and throat cultures from fifty diseased individuals with an
equal- number from normal persons was of interest. The cultures
from persons with infections including scarlet fever, tonsillitis
and "sore throat" were nearly all members of Group A with a
few Group C and G and unclassified strains. On the other hand
less than a third of the cultures from normal throats fell in
Group A, about a third fell in Groups C and G and the rest were
in Groups B or H or were unclassifiable. Hare (1935) in England
found that 63 of 96 cultures from normal throats fell in Group A,
28 in Groups C and G and five in Group B; Davis and Guzdar
(1936) in China reported that 28 of 78 belonged to Group A and
the rest to Groups C and G; Foote, et al. (1936) in this country702 GUNNISON, LUXEN, CUMMINGS AND MARSHALL
found only Group A; Kodama, et al. (1938) in Japan isolated 40
Group A, one Group B, 30 Group C, seven Group F, and 28
Group G cultures; and Kobayashi (1939) in Japan found Groups
A, C, F and G.
The distribution of serologic groups of streptococci in human
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beings seems to be fairly uniform in various parts of the world
as far as is shown by the available data from England (Hare, 1935;
Hare and Maxted, 1935; Congdon, 1935; Colebrook, Maxted
and Johns, 1935), China (Davis and Guzdar, 1936), Japan
(Kodama, et al., 1938; Kobayashi, 1939), Australia (Butler,
1938), the Atlantic coast of the United States (Lancefield, 1933;
Coffey, 1938; Plummer, 1935; Foote, et al., 1936; Rolfs, Trussell
and Plass, 1938), and now the Pacific Coast.
In milk, Group B streptococci predominated, but all groups
except F were found. These findings will be discussed in another
paper.
The results of the various biochemical reactions agreed in
general with those reported by other investigators as summarized
by Sherman (1937), although a few exceptions were observed.
The widely used trehalose and sorbitol fermentation tests
were of definite value in separating the animal strains of Group C
from otherwise similar cultures of human origin belonging to
Groups A, C and, G. No Group A strains which fermented
sorbitol were found although such strains have been described
by Lancefield and Hare (1935) and by Davis and Guzdar (1936).
A number of investigators (Evans and Verder, 1938; Edwards,
1932) have shown that the trehalose and sorbitol tests subdivide
the Lancefield Group C into three distinct divisions of epi-
demiologic significance.
Group B has been tentatively divided by Brown (1939) into
three varieties based on the fermentation of salicin and lactose;
i.e., salicin + lactose +, salicin - lactose +, and salicin + lactose - .
Representatives of each of these varieties were found. The few
salicin + lactose - Group B strains were found only in human
infections as previously reported by Brown. The single salicin -
lactose + Group B culture of human origin was isolated from
urine. The only previous record of such cultures from humanDIFFERENTIATION OF HEMOLYTIC STREPTOCOCCI 703
sources is that of Plummer (1934) who found salicin - lactose+
strains which were later identified as Group B (Brown, 1939)
in the throats of six children. It is as yet uncertain whether or
not these subdivisions represent separate species within Group B.
Unless such variations eventually show definite correlations with
serologic or epidemiologic data it would seem unwise to consider
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them as significant entities.
One of the greatest sources of confusion in work on streptococci
has been the tendency to give species names to strains which
varied from closely related organisms only in their behavior in
a certain biochemical test. The Lancefield classification has
served to overcome much of this difficulty and it would be un-
fortunate to split up the various groups again except in those
instances where the subdivisions seem valid as in Group C.
On the other hand, it would seem unjustifiable to accept the
serologic classification as final and to abandon other tests which
may afford additional significant information as to important
divisions within the groups. It would be wiser to continue to
collect such data but to refrain from assigning the various cul-
tures to definite species in the present state of our knowledge.
Sherman (1937, 1938) has urged that glycerol and raffinose be
included in the list of test substances because little information
has been made available concerning the fermentation of these
carbohydrates since the advent of the Lancefield technic. The
only streptococci of this collection which fermented raffinose were
the minute varieties of Group G and certain Group H cultures.
Inasmuch as these cultures could be differentiated readily by
other means the use of raffinose appears to be of little importance
for routine work. The reactions in glycerol seemed to be of
little or no differential value for the cultures in this series. The
studies of Edwards (1932) and of Niven (unpublished, quoted
by Sherman (1937)) indicated that Group C streptococci of
human origin fermented glycerol but none of those in this collec-
tion did so. Likewise, mannitol fermentation was of negligible
differential importance.
Tests for the curdling of milk gave some information in addition
to the lactose fermentation test but it is doubtful whether this704 GUNNISON, LUXEN, CUMMINGS AND MARSHALL
reaction merits inclusion in routine studies. Hydrolysis of
sodium hippurate on the other hand afforded a reliable identifica-
tion of Group B cultures with but few exceptions. The isolation
from milk of Group A streptococci which split this substance
is of interest.
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As Sherman has pointed out, tests for soluble hemolysin have
little justification because Todd (1934) and others have shown
that under certain conditions streptococci of all groups may
produce this substance. Under the conditions used in the present
experiments some degree of hemolysis was shown by all groups
except D and H.
Sherman (1937, 1938) has suggested that further knowledge of
the action on starch of Streptococcus pyogenes and of biochemically
similar cultures of Groups C and G might be an aid in their
differentiation. All the Group C cultures of animal origin in
this series hydrolyzed starch whereas no Group C or G strains
of human origin did so. However, inasmuch as about one-third
of the cultures of S. pyogenes split starch, the test could not be
used to differentiate this organism from Groups C and G.
It seems evident that the group precipitin reaction is the only
test which, by itself, will serve to identify hemolytic streptococci
with a reasonable degree of accuracy at the present time. Va-
rious combinations of the biochemical tests herein described and
others not included in this report can be used for preliminary
classification but no trustworthy method other than the serologic
technic has thus far been devised that is applicable to all groups.
A number of abbreviated methods have been suggested, notably
the use of sorbitol and sodium hippurate for identification of
streptococci from milk as advocated by Brown (1937). There
are pitfalls in all such short cuts, however, because atypical
strains are fairly common.
The use of the group precipitin test for routine work seems
feasible inasmuch as antiserum for the various groups should
soon be available commercially. By using either a one-tube
macroscopic set-up or the microscopic method the amount of
serum used is very small. The time required for such tests is
less than that demanded for a number of biochemical reactions.
Both the macroscopic and microscopic methods have certainDIFFERENTIATION OF HEMOLYTIC STREPTOCOCCI 705
advantages and disadvantages. The microscopic technic of
Brown requires a minimum of antiserum and of equipment, but
the time involved in reading the tests is greater. More experi-
ence is required in reading the microscopic reactions and false
positives sometimes occur. The macroscopic test requires more
antiserum and antigen and cross-reactions are more common.
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The choice of method should be based on personal preference
and experience.
Although the precipitin reaction supplemented by a few
biochemical tests should suffice for routine studies, research
workers would be wise to heed Sherman's plea that all old tests
should not be abandoned in favor of new ones. Reactions which
were not of differential value among the cultures of this collec-
tion might be of importance among other strains. Wider ap-
plication of some of the classical methods as correlated with the
serologic and epidemiologic data may do much to widen our
knowledge of the hemolytic streptococci.
As Bliss, Long and Feinstone (1938) have said, the grouping of
streptococci may have an important bearing on the use of sul-
fanilamide therapy because some groups are not susceptible
to the action of the drug and some are not sufficiently pathogenic
to warrant its use. Evidence is accumulating which indicates
that these differences are associated with differences in serologic
and biochemical reactions.
SUMMARY
A collection of 561 cultures of hemolytic streptococci from
various sources on the Pacific Coast was studied by the group
precipitin reaction and by certain biochemical tests.
Of 241 cultures from human sources 156 fell in Group A, 13 in
Group B, nine in Group C, six in Group D, one in Group F, 28
in Group G, three in Group H, and 25 failed to react with any
antiserum. Of 188 strains associated with infections, 142 were
Group A; of 53 cultures not so associated only 14 were Group A.
The majority of cultures not of Group A came from the throat
or genito-urinary tract, usually with little evidence that they were
responsible for infection.
Of 320 cultures from animal sources most of which were iso-706 GUNNISON, LUXEN, CUMMINGS AND MARSHALL
lated from milk, 12 belonged to Group A, 263 to Group B, 15 to
Group C, two to Group D, four to Group E, 10 to Group G, one
to Group H, and 13 could not be classified.
The distribution of groups in the vicinity of San Francisco did
not appear to vary greatly from the distribution reported in
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other parts of the world.
It was impossible to identify the various groups of hemolytic
streptococci by means of any combination of the biochemical
reactions studied. Tests for fermentation of sorbitol, trehalose,
salicin, lactose and possibly raffinose and for hydrolysis of sodium
hippurate were of value in preliminary differentiation and in
identification of certain sub-groups. Tests for fermentation of
glycerol and mannitol, curdling of milk, hydrolysis of starch
and production of soluble hemolysin were of little or no differen-
tial value for the cultures investigated.
Double zones of hemolysis in blood plates were formed by ap-
proximately half of the Group B cultures and were not produced
by any cultures of other groups.
The group-precipitin test, using either a one-tube modification
of the original Lancefield technic or the microscopic method of
Brown, is a reliable, simple and practical procedure for the routine
identification of hemolytic streptococci which possesses far
greater accuracy than any other test or group of tests thus far
devised.
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