WTHS

 

THE SOURCE OF STREPTOCOCCI
FOUNDIN
CHLORINATED swmmwc POOLS

Thea-sis for the Degmc cs! M. 5
‘William B. Ardrey
1936

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THE SOURCE OE STREETUJJGGI EQURD IR
CJLORIIEATE DIILLILL: EUOLS

ri‘hesis for Degree of 121.5.
William B. gdrey
1956

LLI CHI GAE 8 11A TE 0 OLLE G;

2"

AGJIUULL‘U RE Hull) hPPLIED 53117110;

THE. SJJRCE 01‘ SEE 1313900000]. FOUND Iii
Cd L011 RATED 51113.1le G P OOLS

A Thesis
Submitted to the Graduate Faculty

For the Master of Science Degree

Department of Bacteriology and Hygiene

by

William B. Ardrey
East LansinngMich.
1956

ACKNCWLEDGEMENT

The writer wishes to thank Dr. W.Lx Mallmann
under whose direction and guidence this work was
carried out and Mr; C.S. Bryan for his many

helpful suggestions.

CWTWTS

ACKNOWLEDGEKEEE
INTRODUCTION

HISTORICAL

PREEARATIGH OE MATERIAIS
EXPERIMEN TAL AND DISCUSSION

I Isolation of streptococci from different regions
of.the body

II Quantitative determinations of streptococci in the
mouth.and in fecal.material

III Controlled exPernnents with swimming pool water to
determine the source of the streptococci

IV’ Fermentation reactions as a means of determining
the source of streptocarci found in swimming pools

SUMMER!
COHOLUSIOHS
LITERATURE CITED

114 TR "U CTI ON

With the introduction of the sodium thiosulphate treated
sample bottle, the presence of bacteria has been no ted in
chlorinated swimming pools that were formerly believed to be
bacteria free. Not only was the colon group noted, but also
the presence of streptococ ci. These latter organisms may
or may not have added signiiim nee over the colon group
index, depend ing upon their source. If they are entirely
of intestinal origin, then they have no sanitary value, as
the colon group of 0335611181118 is ample evidence of intestinal
pollution, but ii‘ thev are or" mouth and nose origin, they
may be of value as indication of respiratory diseases.
Certainly their absence in a pool would indicate the reedom
of the pool water of respiratory disease organisms.

Since streptococci, as demonstrated by LLallmann (1),
do not tend to increase in number after being introduced
into swimming pool water, whereas colon bacilli do, it has
been thought that they might lend valuable aid in determining
the true contamination of the pools. However, most of this
work originated in the analysis of drinking water where
pollution would invariably come i‘rom sewage pollution and

where, therefore, streptococci might be expected to present
a true picture of the fecal contamination. At first it was

not realized that this 'ame picture might not hold good

U]

for swimming pool a.

2

The possibility of large numbers of fecal strep toccc ci
entering the pool would be very slight, as the chance of the
water in the pool becoming polluted by the fecal discharges .
of the bath :rs would be very rare. The only other method
by which fecal streptococci might gain entrance to the water
would be along with small particles of fecal material which
were adhering to the s urface of the body. Even this
possibility would, in some measure, be done away with by
t1 e fact that bathers are required to take a shower before
entering the pool. It is known that the nose and mouth
are the source of large numbers of streptoccnci, which would
have ready access to the pool. During the period of bathing
the mouths and noses of the majority of bathers would be
washed out with water several times, each time carrying
large numbers of streptococci into the pool. It would seem,
therefore, that the chance of the pool becoming contaminated
with streptococci from the nose and mouth would be greater
than the chance of contamination from fecal material.

Manheimer (2), in a review of the literature on enteric
and reapiratory diseases obtained through bathing, points
out the fact that while there have been a great many enteric
diseases transmitted in this manner, t‘ey have all resulted
from bathing in rivers or in pools where the water is

untreated. 0n the other hand, those cases of reapiratory

5

diseases which have been obtained while bathing were mostly
of the type caused by pyogenic cocci and were obtained from
swimming pools which were apparently free from intestinal
pollution. Grierson (3), in his review of the literature
came to much the same conclusion as Iian’heimer, namely, that
though the transmission of enteric diseases may be of
importance in the case of sewage polluted waters, it is the
reSpiratory diseases which are of prime importance in
swimming pools. Hasty (4) states that ear and reSpiratory
diseases are oftrn transmitted by swimming pool water, and
demonstrates the fact that water gets into the nasal
chambers of the Iriajority of swimmers, carrying with it
whatever sort of contamination happens to be present.

Although investigators seem to disagree as to the
source and importance of these streptococci found in swimming
pools, no reference is made of anyone trying to determine
from which source the greatest number are derived. Since
many different diseases such as conjunctivitis, otitis media,
sinusitis, and many other sinus and respiratory diseases
are sometimes caused by certain pyogenic cocci, it is of
some importance to try to discs! er the actual source of these
streptococci found in swimming pools. It is, therefore,
the purpose of this paper to demonstrate whethe~ these
streptococci have as their source the nose and mouth or

fees 1 ma terial .

4

HISTARIQAL

 

Since our entire present method of swimming pool
analysis is based upon the method used for the analysis
of drinking water, it is not surprising that most of our
ideas concerning streptococci found in swimming pools comes
from the same source. Prescott and ‘iiinslow (5) report that
Houston in Thigland, in 1900, was the first to emphasize
the importance of streptococci found in water. He pointed
out that the streptococci were characteristic of animal
discharges and were, therefore, quite indicative of dangerous
pollution. The first work: in this country was carried out
by Winslow and Hun-newell (6), in 1902, when they isolated
streptococci along with Escherichia 0011 from the hands of
school children. Later they found them to be present in
sewage, feces, and in water polluted by sewage. In 1904,
Prescott and Baker ('7) showed these organisms to be present
in each of 50 samples of polluted water, whereas they could
not be demonstrated in nonpolluted waters. Savage and Read
(8), in 1916, report that the estimation of streptococci
in water is of undoubted value as evidence either for or
against excretal contamination. Following this a great
deal more work was done along the same line by other investi-
gators, ineach case the streptococci being found along with
_E_1_s_g_1_1. £91; in the sewage polluted water. It is not surprising

that when streptococci were first demonstrated to be present

5
in swimming pool water they were assumed to be of fecal

OI‘ igin.

Hasty (4), in 1937, called attention to the fact that
the water in a swimming pool during the periods of its use
would be subjected to the tasiings from both the nose and
mouth of the bathers, thus receiving large numbers of strepto-
cocci which might be important in the transmission of disease.
Liallmann (l), in 1928, was the first to use streptococci
as an indicator of swimming pool pollution. is states,
"Streptococci are constant indicators for intestinal pollution
and the number found in the pool parallels the amount of
pollution as indicated by the number of bathers." Grierson
(5), in Speaking of the method 3 used for determining the
pollution of swimming pools states, "It is customary, there-
fore, to access results on the presence or absence of
Bacillus coli in various quantities of water, taking into
consideration the total bacterial count and the presence
or absence of faecal streptococci and other organisms."
Horwood, Gould, and Schwachman (9) were of the Opinion that
the streptococci found in swimming pools are of little
importance in the determination of fecal pollution, but
may be a very important factor in the transniss ion of
diseases of the nose and throat. In all the cases where

streptococci have been used as a confirmator; test for

intestinal pollution of swimming pools it has been assumed
that they were of fecal origin, but there is no reference

in the literature where eXperiments were performed to
demonstrate this. In the cases winre investigators believed
the streptococci to have their origin, at least in part,

in the nose and mouth, there has been n work done to confirm

this belief.

rereammon pg LLAMI..;..I£-.-LS

 

 

In deteimining the presence of streptococci from
different parts of the body standard beef infusion broth
was used. Its reaction was adjusted to between a pH of 7.4
and 7.6. To this broth was added 5 per cent of sterile
bovine blood. In order that dilution determinations might
be obtained, in the case of fecal material and material
taken from the mouth, this broth was thm tubed aseptically
in 9 0.0. amounts. ’i‘hese tubes were then incubated fora
period of twenty-four hours at 37°C. in order to determine

if there was any contamination.

In the isolation of pure cultures of streptoccnci and
in the making of quantitative counts blood agar was used.
A base medium of beef liver infusion agar was used, as
recommended by stafseth (10). The reaction was adjusted

to a pH of 7.4 to 7.6. The agar was then placed in flasks

7
in 200 0.0. amounts. In the preparation of the plates
the agar was nelted and after being cooled t3 between 47
and 50° C. 5 per cent of sterile blood was added and the
agar then poured into plates. This same blood agar was
used in the preparation of sLnts for “die carrying of pure
cultures. The tubes, before being inoculated, were incubated
at 57°C. for 24 hours to determine the presence of cantamina-
tion.

In the isolation of pure streptococci colonies frmu
different parts of the'body, an.attempt was made to use
gentian-violot, as recommended by Bryan (11),£;s an inhibiti ng
agent for the other organisms present. Strep‘ococci are, for
the most part, fairly resistant to the action of germicides,
capacially that of certain dyes. Norton and Davis (12),
in their work on the bacteriostatic action of dyes found
that most streptocazci are inhibited in their growth by
gentian-violet in a dilution of 1 to 40,000. However, in
dilutions of 1 to 150,000 and 1 to 200,000 strep;ococci
are not inhibited whereas a great many other organisms are.
Therefore, in the prep;ration of plates for the isolation of
streptococci colonies, one per cent aqueous gentian-violet
was added in amounts of about 0.1 0.0. per 200 0.0. of agar.
Five per cert of blood was then added and the plates poured.
In many cases, however, such small amounts of agar and dye
were used that it was rather difficult to obtain the rigit

dilution. As a result of fliis the streptococci and other

8

organisms were either both inhibited or the streptococci
colonies were completely overgrown by the other organisms
present. because of this variability in inhibitory action,
gentian-violet was added only to those plates where strepto-
cocci were being isolated from feces and which in the absence
of an inhibitory agent, were overgrorm in almost every case.

Sample bottles for the collect ion of water from swimming
pools were prepared as recommended by hallmann and Gary (15),
with the following exceptions. Instead of the addition of a
small crystal. to a moist bottle or 0.5 cc. to a dry bottle
and sterilization by moist heat, pondered sodium thiosulphate
was added to dry bottles and these were sterilized in the
hot air sterilizer instead of in the autoclave, (Devereux
and hallmann (14).

The carbohydrate media used in the fermentation tests
were prepared by adding one per cent of the carbohydrate
to peptone water. The peptone water was prepared by adding
10 grams of peptone and 5 grams of salt per liter of mater
and the reaction adjusted to a pH of 7.4. To this was then

added 2 0.0. of lindrade's indicator per liter of solution.

QWML
Egperiment :_[_

Since the bathers themselves are the only conceivable
source by which streztococci might gain entrance to swimming
pools, a study was made of different surfaces of the body,
the mouth and fecal material in order to determine which

of these sources would be the greatest contributing factor

for these streptococci. Along; with this, pure cultures
were isolated from these different sources for comparison
with pure strains isolated from swimming pools. For this
work liver infusion blood agar was used. The organisms were
classified into the three types of Smith and Brown (15).

In the determination of streptococci on the surface
of different regions of the body sterile cotton swabs were
used. These were prepared and placed in tubes containing
1 0.0. of sterile distilled water. These swabs were rubbed
over different surfaces of the body. In order to cbtain
comparable results shares of about 1 1/2 to 2 inches square
was swabbed. ‘l‘he swabs were then placed in tubes of broth
containing 5 per cent blood and were incubated at 37°C. for
a period of 24 hours. At the end of this time a hanging drOp
was made of each culture, and if the presence Ci“ streptococci
was noted, smears were made and stained by Gram's method
in order to confirm their presence. The regions from which
these different swabs were taken are as follows: mouth,
anus, abdomen, arm (upper), arm (lover), ear, nose (internal),
hands, chest, thigh, leg (lower), face, back, ankle, and
fecal material. The results of this ex;eriment are shown
in Table I. '

In cultures made from the mouth and frcm fecal material,
as might well be expected, the presence of streptoccn ci was

noted in almost every instance. The nly exceptions to this

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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10
were samples No.3 and No.15, obtained from fecal material.
Eve: in the case of L0. 3 there were a number of chain-like
organisms present which had the appearance of very short reds.
If these had been transferred a number of tnnes and a more
detailed study made of them they migh have been.proved to be
streptococci. Sixteen cultures out of 25 from the nose
proved to have streptococci present in them. This was also
to be espected as the mucous membranes of the nose like
those of the mouth and threat are considered as one of the
chief sources for streptocazci. Samples from the anus showed
sixteen out of the twenty-three cases to have streptococci
present. This large number of cases is beyond doubt due
to Contamination with fecal material. Samples from the
thigh show eight of the cases to have streptococci present
whereas there were only two cases where streptococci could
be demonstrated on the lower section of the leg. dare again
those cases found on the thigh.may have been due,at least in
part, to contamination with.fecal.material. Samples from
the hands showed streptococci in ten out of twenty-three
cases. This number is probably due to the fact that the
hands are very often brought in contact with the nose and
mouth and would thus tend to become contaminated with
streptococci from these sources. For the most part the
rest of the regions studied tended to show none or a very
few cases with streptococci. The ear and face each showed
five cases to have streptococci present, but all the other

regions examined were positive in no more than three cases

11

and in several instances in no case. From these results
it would appear that the two chief sources from which
streptococci might gain entrance to aid hurling pools would
be from the nose and mouth of the bathers and possibly frcm
small particles of fecallmlterial adhering to the'body.
The other body surfaces would seem to be of very little
significance in raising the nul'lber of streptococ ci in the
pool. In the first place streptococci are present in such
few cases that even if they were introduced into the pool
they would have little effect in increasing the total
number present. Then there is the fact that in all well
controlled pools the bathers are required to take a bath
before entering the pool. This muld tend to wash off a
goodly number of those present so that in most cases there
would be little danger of the pool becoming contaminated
from this source.

In attempting to isolate pure cultures of streptocazci,
steri‘e swabs were first tried. These were dipped into the
tubes of blood broth; vhich had previously been inoculated with I
fecal.material, material taken from thelnouth, and from
swimming pools; and were thm streaked on blood a3ar plates.
This method, however, did not prove to be very successful,
as the other organisms usually outgrew and overgrew the
colonies of streptococci. an attempt was then made using

dilutions of the inoculated broth and plating; inblood agar.

12

rl‘fhis method proved to be more successful except that in
some of the cases, where fecal material was used, the other
colonies would still overgrow the streptococci colonies

in the dilutions where the streptococci were present. It
was found, hoa-zever, that by the addition of gentian-violet
in a dilution of about I to 150,000 that these organisms
could be inhibited to such an extent that colonies of
streptococci might be obtained. .Jhen typical streptococci
colonies were noted the y were fished with a platinum needle
and streaked on blood agar plates, where they were incubated
at 57°C. for 24. hours. nt the end of this period hanging
dropswere made of the colonies, and, if the presence of
streptococci was noted, it was confirmed by stained
preparations. The colonies were the; transferred to blood
agar s12 nts where they were transplanted every three weeks

to insure good growth.

EXpe rimen t I;

illce most of the streptococci in the sv-Jinuuing pools

U1

Seem to be of either mouth or fecal origin an attcn‘lpt was
made to determine the number of streptococci present in
certain given amounts of material from each of these sources.
In making these quantitative tests for the number of
streptococci in the mouth, 10 c.c. of sterile water was
taken into the mouth and washed about and gargled for a
period of about half a minute. This was then diluted out

into tubes containing 9 c.c. of blood broth in dilutions

13

of l to 10, l to 100, 1 to 1,000, l to 10,000, aid 1 to
100,000. 'l‘hese tubes were then incubated for a period of
24 hours at 57°C. at the end of this time the presence
of streptococci was determined by making 831108.138 of each
dilution and staining; by Gram's method. The results of
this work are shown in Table II.

In only two of the cases Nos 9 and 21, were. there no
streptococci present. In case No.17 there were only a
hundred streptococci obtained in the one washing. I'Jith the
exception of these three cases all showed streptococci to
be present in numbers between 1,000 and 1,000,000. The
average number ob tained with one washing was about 100,000.
This, however, does not mean that this would be al’l;"‘-..l'..’1'le-re
near the total number of streptococci entering the pool
frcm the mouth of am,r one bather. In cases where the
mouth was washed a second time innaedia tely following the
first washing it vas found that relatively high counts could
still be obtained.

In order to checl: on this dilution method as a means for
deternnning; the nunber of streptococci, samples 1 to '7 and
18 to 20 inclusive were also determined by the plate method.
In this work dilutions of the samples were made the same
as before; 1 c.c. being placed in sterile petri dishes.

Liver infusion blood agar was then added and the plates

incubated for 24; hours. nt the end of this time the number

TABLE II
Streptococci in.Louth

 

‘ Dilutions 'Numher Obtained
NO.'lleOlelLOOQ'1:10.000Tl:100,000'1:1,OOOLOOO'in One fla§iiggg
I I

l ' + ' + ' + ' + ' — ' 100,000
2 I * I + I - I - I - I 1,000
3 I I . I I _ I _ I
4' i ' i ' i ' + ' - ' 13.23833
5 I + I + I + l + I I ’
' ' ' ' ' + ' 1,000,000
.6 + + + + + 1,000,000
4. 1
7 I I + I + I _ I _ I 10,000
8 ' + ' + ' - ' - ' - ' 1,000
9 I _ I _ I _ I _ I H I O
10 ' 'I ' + ' '+ ' - ' - ' 10 000
11 ‘ + ' + ' + ' - ' — ' 10'000
12 ' + ' + ' - ' - ' - ' 1’000
15 : + : + : + 1 + : + I 1,000:OOO
14 + + + + - ' 100 000
15 I + I + I + I ' , I .0
16 I + I I ' - ' + 10,000
, , + + + - ' 1,000,000
17 + ” ' ' ' - ' - ' 100
18 ' + ' + ' + . . .
. + - 100 000
19 l + I + I I I 3 ’ A
20 ' + ' + ' - I - I - I 1’000
21 , , , + ' * ' ~ ' 10,000
~ - - - - O
22 ' + ' + ' + ' + ' - ' 100 000
25 ' + i ‘I' ' + ' + ' _ I 100’0\
24 ' + ' + ' + ' ' I - ’AYO
05 I I . , + I “ l,OOO,UUO
0 , + , + , + - ' - 1 10,000

1‘

 

14
of streptococci colonies were determined by their action
on the blood gar. The results of fiiisinethmi are shown
in Table III.

In ahaost every case the results were similar to those
of the dilution method, except that the number obtained by
plate method, as might be eXpected, was slightly higher than
by the dilution method. Emples Nos. 2 and a, as determined
by the plate method, showed no streptococci to be present,
whereas the same samples tested by the dilution method
skwwed sample No.2 to have 1,000 streptococci p?esent and
sample No. 5 to have 10,000 streptococci present. In the
plate method the lack of streptncncci in these twa instances
was probably due to the fact that they occurred only in
dilutions which wouhi'be overgrown y the other types of
bacteria present.

In determining the number of streptococci p?esent in
fecal material much the same method was used as in the
determination for these in the mouth. Samples of feces
were obtained from different individuals and were weighed
out in one gram amounts into large sterile test tubes.

Ten c.c. of sterile water was then added an; the material
well mixed. This was then diluted in tubes of blood broth
in dilutions of 1 to 10, l to 100, l to 1,000, l to 10,000,
and 1 to 100,000. These were incubated as efore and the
determination for streptcmncci made by making smears and
staining with.Gram's stain. The results of this examination

are shown in Table IV.

TABLE III
Stre tococci in Mouth

 

 

 

 

' Dilutions 'Number Obtained

Ho ' 1'10 ' :100 ' 1:1000 '1;1Q$200 '1:102£00071n One Washing__

1 'overgrown'overgrownfovergrown' 5 ' - ' 300,000
I I I I I I

2 I - I - I - I - I - I O
I I I I I I

3 I - I - I — I - I - I O
I I I I I I

4 'overgrownzovergrownfovergrown: 2 ' - ' 200,000
I I I I

5 ' " ' " ' " ' 9 ' - ' 900,000
I I I I l I

6 a " 1 " 1 " :overgrown' 2 : 2,000,000

I

7 ' " ’ " ' 8 ' - ' - ' 80,000
I I I I I I

18 I II I II I 6 I _ I _ I 60, 000
I I I I I I

19 I II I II I 4 I _ I _ I 4:0, 000
I I I I I I

20 I II I II I 2 I _ I _ l 20. 000

 

TABLE IV
Streptococci in Feces

 

1 Dilutions "Ihnnber Obtained

 

NQLT1:100'lleOO:1:10,000:l:IOO,OOO:1:1,OOO,OOO' In One Gram
I “r .

I
1 I -+ I + I + I _ I _ I 10.000
I I I I I I
2 ' + ' + ' + ' - ' Q ' 10,000
I I I I I I
3 ' + ' + ' + ' + ' - ' 100,000
I I I I I I
4 I + I + ' + ' - ' - ' 10,000
I I I I I I
5 I + ' + ' + ' - ' - ' 10,000
I I I I I I
6 I 4- I 4- I I- I - I - I 1,000
I I I I I I
7 I + I + I + I I. I - I 10’ 000
I I I I I I
8 : + : + : + 1 + : - ' 100,000
I I I I I I .
9 , + , + , - , - , - , 1,000
I I I I I I ‘
10 I ' I " I " 'I " I " I O
I I I I I I
ll , + , + , + , + I + , 1,000,000
I I I I I I ,
12 , + , + , + , + , - , 100,000
I I I I I I
15 , + , + , + , - , — , 10,000
14 : + 1 + : + : + : - : 100,000
I I I I I I
15 , + I «r , - , - , - , 1,000
16 ', + I + ', + ', — ,' - ', 10,000
I I I I I I
17 I + I + I + I " I - I 109 000
I I I I I I
18 , + , + , + , - , - , 10,000
I I I I I I
19 , + , + , + , + , + ' 1,000,000
I I I I I I
20 , + , + , + , + , + , 1,000,000
I I I I I I
21 , + , + , - , ~ , - , 1,000
I I I I I I
22 I + I ‘ I ' I ' I ' I 100
I I I I I I
25 , + , + , + , + , - , 100,000
I I I I I I
24 , + , + , + , - , - , 10,000
I I I I I I
25 I + I + I + I " I '-' I 10, 000

15
In this work: there was only one case, $0.10, which
showed a cdnplete absence of streptococci. Case 150.22 showed
only 100 streptococci to be present per gram of feces whereas
in all the rest of the cases the numbers ranged between
1,000 and 1,000,000. Illhe average numb er per gram of fecal

material was about 10,000.

Although no direct comparisons cm be made between the
number of streptococci ch tained from a washing of the mouth
and from a gram of fecal material, these experimems at
least show that either one of these sources would present
a very plentiful supp y of streptococci if bro ught into
contact with the water of a swimling pool. 0n the whole,
it would seem that the chance of swimming pool water be-
ccming polluted by fecal discharges of the bathers would
be very rare. At least it seems safe to say tlat it would
be of such rare occurrence that it would play no @rt
in the day to day high streptococci indices which may be
obtained from almos t any swimming pool. liven smll
particles of fecal material adhering to the body would be
removed in large measure due to the fact that bathers are
required to take a bath before entering the pool. 0n the
other hand it is easy to see how the large numbers of
streptococci contained in the nose and mouth would have
ready access to the water of the pool. Even though spitting
is prohibited in the pool, there is little doubt but that
the nose and mouth of each bath-er is well washed out
several times uuri 113 the course of 8‘5111111311'15; each

washing carrying large numbers of streptococci into the pool
V o

16

linger im an t I II

 

In this series of experiments an attempt was made to
determine whether the streptococci found in swimming pools
were of oral or fecal origin. These ex-perimmts were con-
ducted with a class of forty students in one of the Junior
high school pools. rIlhe residual chlorine in the pool was
first determined by means of the ortho-tolidine test and
control samples of the water in the pool were taken in
sodium thiosulphate treated sample bottles before the class
was allowed to enter the pool. All the samples taken,
includ ing the controls, were taken in triplicate; one
at each side of the pool and one at the shallow end. The
three samples were taken simultaneously. After these three
control samples had been takm the class was requested
to enter the pool at the shallow end, and, in so doing,
to be careful that their heads were continually above the
water. They were then asked to move about, meanwhile keeping
their heads well out of the water in cr der that there might
be no chance of streptococci from their mouths entering the
pool. At stated intervals sataples were taken of the pool
water from the three different positions. After several
samples had been taken in this manner, the class was givai
sane breathing exercisa which entailed repeated total

submersion. In a great many of the cases this had the

effect of introducing water into the nose and mouth, frcm

l? i
which it was shortly returned to the pool. As before
samples were taken at regular intervals.

These samples were then carried to the laboratory
where 10 c.c. of each sample was pipetted into each of five
tubes of double strength lactose broth and l c.c. into each
of five tubes of single strength hctose broth. These were
incubated for a period of 48 hours. One c.c. portions of
the water were plated out in plain agar and counts of the
I'lumber of bacteria present were made at the end of 24 hours.
At the end of 48 hours the tubes of lactose broth were
examined and those showing gas were streaked on eosin-
methylene blue agar to ccnfirm for §_s_c_}r_1_. 991;. The. presence
of streptococci was first determined macros00pical 1y by
the method suggested by Liallmann and Cary (15). This
consists of holding the tubes up to the light and looking
for a granular precipitate. If this precipitate was present
after the tubes had stood for ’72 hours, the supernatant
liquid was drawn off and smears were made of the precipitate.
These were then stained by Gram's method and examined
. microscopically to confirm for the presence of streptococci.
From these results indices for the gag}. p__o_l__i_ and the strepto-
cocci present in the pool were obtained. The results of
this experiment are found in Tables V, to Li inclusive. Each
Table is divided up into three parts A, b, and C; repre-
sent ing- the different positions about the pool from which

the samples were taken. The number of bathers and the parts

TABLE X
Streptococ cus and Colon Index

P.P.M. of Chlorine -- 1
Number of Bathers -- 4-0

 

 

 

 

 

 

 

 

 

 

A
Ncr th Side of Pool
Sample Time Streptoco: cus Colon Bacterial
number in pool ind ex index count
1 O - - 19 Control
2 1 min. " - 56
Body
4 5 nine 4 'l' 60
5 4 min. 4 - 19
B
South Side of Pool
Sample Time Streptococcus Colon Bacterial
number in pool index ind ex count
5 l O - - 48 Control
2 l min. - - 8
Body
4 5 min. 2 2 91
5 4 “lino 2 2 166

TABLE 1 (continued)

Streptococcus and Colon Index

V C
End of Pool

 

 

 

Sample Time Streptocarcus Colon Bacterial
number in pool index index count
1 O - - 24 Control
2 l min. "' - 97
5 2 min. 2 6 90
Body
4 3 min. 4 - 53
5 4 min. 2 - 3

?OPOIIIO
Emmer

TABLE i1

Streptococcus and Colon Index

of Chlorine -- .6
of Bathers -- 40

A

North.Side of Pool

 

 

 

 

 

 

Sample Tune Streptocoicus Colon Bacterial
number in pool index index count
1 O - - 7 Control
2 l min. - - 15
5 2 min. - - 29
4 5 min. - - 482 Body
5 4 min. - - 55
6 5 min. - - 26
B
South Side of Pool
Sample Time Streptococcus Colon Bacterial
number “in_pool index index count
1 0 - - 6 Control
2 l min. 4 4 ll
5 2 min. - - 51
4 5 min. - 4 28 Body
5 4 min. - - 96
6 5 min. - - 20

TABEE VI (continued)
Streptococcus and Colon Index

C
End of Pool

 

 

 

 

 

Sample Time Streptocarcus Colon Bacterial
number in p991 ,g_, index index count
1 O __ 4 - 5778 Control
5 2 min. - - 5650 Body
4 3 min. - — 4509
5 5 min. 40 6 4794
mouth
6 6 min. 10 - 1705

TABLE VIII
Streptocaacus and Colon Index

P.P.E. of Chlorine -- .75
Rumber of bathers -- 28

 

 

 

 

 

 

 

 

A
Nwth Side of Pool
Sample Time Streptococcus Colon Bacterial
number in pool index index count
1 ___g__QV ‘ ==:_ - 15 Control
2 l min. - - 16
Body
5 2 min. - " 4O
4 4 min. - - 42
5 5 min. 2 - 57 Moufii
6 6 Kline "" " 47
B
South Side of Pool
Sample Time Streptococcus Colon Bacterial
number in pool index index count
1 O - - 26 Control
2 l min. - - 51
Body
5 2 min. "' i - 57
4 4 min. 4 2 81
5 5 min. 6 - 76 heuth
6 6 min. 2 - 49

TnBLE VIII (continued)
Streptococcus and Colon Index

0

End of Pool

 

 

 

Sample Thne Streptococcus Colon Bacterial
number in pool index index count
1 O - - 25 Control
2 1 min. " " 65
Body
5 2 min. - - 57
4 4 min. 4 - 109 I—
5 5 min. - - 56 .houth
6 6 min. 4 - 67

 

 

 

 

 

 

 

TABLE IX
Streptococcus and Colon Index
P.P.L1. of Chlorine -- .5
Number of Bathers -- 58
A
North Side of Pool
Sample Time Streptococcus Colon Bacterial
number in pool index index Count
1 0 - - 29 Control
2 1/2 min. - - 55
Body
3 1 min. 2 - 119
4 2 min. 20 - 96
5 2 1/2 min. - .. 72 Licuth
6 3 min. 20 - 96
B
South Side of Pool
Sample Time Streptoco: cus Colon Bacterial
wber in pool ird ex ind ex count
1 0 - - 16 Control
2 1/2 min. - - 20
Body
3 1 min. - - 44
4 2 min. 6 - 57
5 2 1/2 min. 8 - 77 Liouth
6 5 min. 4 - 91

TABLE 11 (continued)
Streptococcus and Colon Index

C
End of Pool

 

 

 

 

Sample Tune Streptocoecus Colon Bacterial
number in pool index index count
m =======
1 O - - 22 Control
2 1/2 min. 2 - 68
Body
4 2 min. 4 - 58
5 2 1/2 min. 6 - 44 Lieuth

18

per million of chlorine is recorded in each caseat the
top of the Table.

These Tables are rather significant in that in every
case there is a rise in the streptoccc-cus index immediately
or soon after the heads of tin bathers had been submerged
in the water. ‘g‘lhat is still more important is the fact
that in a great many of the cases the index increases any-
where from five to thirty times over that obtained whm
only the body was in contact with the water. This in it-
self would tend to show that some new plentiful source of
streptococci had been intro.; 1:: ed. that this new supply
of streptococci can not be attributed to fecal discharges
of the bathers is shown by the fact that in no case was
there a like increase in the colon index as might be eXpected
if the fecal material was reSponsible. Furthermore the
presence of Each. coli was noted in only five out of the
fifteen series of samples taken, and in only two samples
did the index reach six. This would tend to show that no
large amounts of fecal material had entered the pool.

In order to show that this sudden rise in the strepto-
coccus index following the submersion of the bathers' heads
was not due to the gradual release of the organisms from
the body, although the sudden and in most cases rather
large increase would in itself tend to disprove it, another

set of expewiments was run. In these ei-zperiments the same

l9

procedure was used as before, except that all of the samples
were collected with only the bodies of the bathers expcs ed
to the water and without their heads being submerged at
any time. Th -, results of these experiments are shown in
Tables X to XII inclusive. In none of these cases, where
the mouths of the bathers were not allowed to come in con--
tact with the water, did the streptococcus index rise above
4, whereas in the previous cases, Tables V to IX inclusive,
w‘ ere the mouths of the bathers had been in contact with
the water the index ofte: rose as high as 20 and in one
case, Table VII-C, as high as 60.

In Tables X to £11 inclusive, the streptococcus ind ex
seems to parallel roughly the colon index. This might well
be eXpected due to the fact that here the tm organism
are assumed to come from the same source. However, if
this were true it would be eXpected that somewhat the sans
results wouldshow up in Tablas V to IX inclusive, in those
samples which were taken dun ng the period when only the
body was eXposed to the water. This, however, does not seem
to be the case with the possible exception of Table 'V-A
and C. In no case does there seem to be any relationship
between the streptococcus or colon indices and the total
bacterial count.

Exgr iment I1

Although all of the above results seem to point to the

fact that the large majority of the streptococci found in

swimming pools have the nose and mouth as their sour ce, an

TABLE III (continued)
Streptococcus and Colon Index

C

 

 

 

End of 33001
Sample Time S trep toco ccus Colon bacter is 1
number in pool - index index count
1 O - - 25 Control
2 1/2 min. '— — 119
3 1 min. - - 135
4 1 1/2 min. - 2 894 Body
5 2 min. - - 312
6 2 1/ 2 min. 4 - 153

20

attempt was made ccnf irm these observations. This was done
by taking a number of pure cultures of streptococ-ci,-vhieh
had p'eviOusly been isolated from the mouth, fecal material,
and swimming pools, and testing than with certain carbohy-
drates, to determine their ability to ferment. It was hoged
that in so doing those streptococci which had been isolated
from swimming pools might fall into certain definite groups
which would correSpond with certain groups of reactions
as produced by either those streptococci isolated from the
mouth or from fecal material. In this wcrk the carbohydrates
used were lactose, salicin, mannite, and inulin. The
action on blood agar and in litmus milk was also determined.
In naming the different groups formed, the classification
of Holman was used.

many attempts have been made to classify streptococci
according to their ability to ferment different carbohydrates.
As a result, a great many different test substances have
been used, and a great many different results have been
obtained, thus causing acme workers to doubt the value of
such a procedure. Bergey (16) came to the conclusion that
the action of streptocncci on different carbohydrates was
of little value in the determination of their classification,
due to the fact that they were so prone to die out in the
media, thus making it difficult to retain their vigor.

Thro (17, 18) demonstrated a number of different factors

21

which would tend to produce variation in the results of
fermentation reactions of streptococci. Some of the factors
are as follow; Dry media, faulty preparation of media,
variability of growing power of different strains, length

of time grown on artificial media, complexity of the sub-
stances used and the lad: of pure ones, titration, alkali
frcm glass, and the exceptional variability of this particular
group of organisms. Holman (19),a1though reporting favorable
results, states that confusion may easily arise from the

fact that certain strains may lose their vigor when grown

upon arti ficial media .

water 8 d isagree

vs
V

Aside from thefact that many investi
as to the importance of various carbohydrates in the classi-
fication of streptococci there is another factor which enters
in to make it a rather doubtful procedure to use in deter-
mining the sowce of streptococci found in swimming pools.
According to the present methods of classification, the
great majority of those types of streptococci which are
found in the mouth may also be found in fecal material, and
those types found in fecal material may also be found in
the mouth. The only two varieties which would seem to have
any value at all in a determination of this kind would be
Streptococcus salivarius, which is found largely in the
mouth, and Streptococcus fecalis, which is found for the
mcst part in the contents of the intestine. live: here there

would seem to be nothing which would hinder an intercla nge

22

of sources, thus causing a great deal of confusion in
the kind of determination attempted.

The chief wort: and that of most value along the line
of using carbohydrates in the classification of streptococci
seems to have been done by Gordon, Andrews and Herder,
and Holman. Gordon (20) was the first to maize use of
carbohydrates as a means of classifying:- streptococci. In
this work: he studied thirty-five different compounds and
finally selected as those having the most value; lactose,
saccharose, raffinose, inulin, salicin, coniferin, and
mannite, along with the clotting of litmus mill: and the
reduction of neutral red. This same method was further
developed by nndrews and Border (21), and used as the
basis for the classification devel Oped by :iolman (20).
This classification as deveIOped by Holman is give: in
Table XIII. It was this classification which was used in
naming the different groups as'determined by their fermen-
tation reactions.

In determining the types of colonies on blood agar
the classification * as worm-ed out by Smith and Brown was
used. Schottmuller (25), in 1905, was the first to make
use of blood agar'in the demonstration ‘of streptococci in
regards to the effect of their growth on the red blood cells.
By this method he divided streptococci into two groups.

The: e which dissolved the red blood cells he called

TABLE XIII

Holman's Fermentation Reactions

* oooo....L&ctose.o.....

 

m
H
m
bu
'3
S
(D
U:

- I

° 0

° 0)

O

4' +3

O

(U

’2‘

I

+

JMannit-

+SaIICin-

+Mannit.

+Mannit-

+Mann1t-

 

 

 

 

 

+Salicin-

+Salicin-

+Salicin-

+Salicin- +Salicin-

+Salicin-

+Sa1101n-

Str.

Str.

Str.

Stro

Stro

Str.

Str.

Str.

Str.

Str.
Stro

Str.

Str 0

Store
Stro

Stro

ignarus

equinus

non-hemolyticus iii

non-hemelyticus ii

salivarius

mitis

non-hemelyticus 1

recalls

subacidus

equi
hemolyticus iii

hemolyticus ii

anginosus

pyogenes

hemolyticus i

infrequens

D3
()3

hemolytic streptococci or "Streptococcus pyogenes var.

ersipelatis," and thee e which caused a greenish discoloration

 

of the blood with only slight or no hemolysis of the blood

cells be called "Streptococcus mitior var. viridans." Smith

 

and Brown (15), in 1915, and Brown (24), in 1919, in their
description of streptococci colonies on blood described three
types which they called "alpha," "beta," and "gamma." Their
descriptions of these three types in brief is as follows:

"Type alpha. as observed after forty-eight hours
incubation the change proiuced by streptococci of tiis typa
may be described as a somewhat greenish discoloration and
partial hemolys is of the blood corpuscles 13111196181281};
surrounding the colony forming a rather indefinitely bounded
zone 1-2 m.m. in diameter and surrounded by a second, narrow,
clearer, not discolored, partly hemolmaed zone.

"Type beta. This type of appearance in blood agar may
be described as a colony surrounded by a perfectly clear,
colorless zone of hanoxysis. There is no trace of discolor-
ation and when.viewed miscrOSOOpically no corpuscles are
seen.in the medium.surrounding the colony. The colony
itself is grayish by transnitted light.

"Type gamma. By the gamma type is meant the growth of
the streptococcus colonies within and on the blood agar
plate without the pcoiuction of any perceptible hemolysis
or discoloration of the surrounding medium airing incubation

or refrigeration."

24

The results of this experiment are given in Tables
XIV, i“, and.£VI. Table XVII shows the different groups into
which the strains smidied fell along with their classification
as worked out according to Hohnan. From the results datained

it aeoears, as might be expected, tfiit thiszuethod of

L.

‘

determining the origin of streptococci in swimming pool
water has little value. The two groups, Table XVII~I and V,
which might be eXpected to be of some aid in such a deter-
mination are not outstanding enough to be of much value.

As pointed out before these two strains, Str. salivarius

 

 

1

and Str. fecalis, are fairly characteristic of the mouth
and of feca1.material respectively as their names wouhi
imply. However, like all the other strains it is not

impossible for them to be found in either place. This

J

is fairly well vemonstrated by the results obtained. ” r.

salivarius was found in eight cases from thelnouth and in

three cases from fecal material. Str. fecalis was found in

 

 

no case from the mouth and in ten cases from fecal.material.
Str. salivarius was found to be present in the swimming pool
water in five cases and §§£. fecalis in two cases. This

is more or less the relationship which might be eXpected
judging from the previous eXperiments, but there is not
nearly enough material from which to draw any definite

ccnclusions. The other eight groups into which the

streptococci from these these different saurces fell are

7-- --fi
.D ' H

(1»

XIV

Fermentation Reactions

'outh

’
- -n.

n
.23

4D

UR

«U
«.D

$Allk
'Acid’Clot

Inulin’T

Salicin‘T Mannite '
I

 

Culture

"""""""""

I II II I. -I II cl cl cl II II I. II I- I-

 

'IIIIIIII'IIII"-

IIII"IIII"

"'IIII"

'l'tl-IIIIIIIIIII

LEXV

TAB
Fermentation Reactions

SOURCE--Eeces

1-1 1k.
'Acid'olot

 

Culture' Type“TWZactose r'Salicin ' hannite—T'lnulin '

II II II cl ll ' II II II II ' II ' I. I II II ' ' II I. ' 'I ' ' ' II II Ql

'
"l'."' "

'II' ' ' ' I I ' t 'I
In I. ' l 'I I. II cl II "' Illl OI "

 

IIIIIIIIIIII'OI"'IIIIQI""QI

[cl-ll." cl
'II" """t'

a a a a a a a a a a a a a a a a a a 8 a a a
h h h h h n. h h n. h n h h h h h h h m h h h m h
D. b. D. D. p p U. D. P p P E. p .0. p p p p p D. p p p p
l l l l l l l l 1 l l l l l l l l l l l l l l l
f A nu A n a. :A A n A: A a L“ A A a a a A A A A A A A.
l 2 3 4. 5 6 7 8 9
O l 2 3 A. 5 6 7 8 9 O l 2 5 4
l l l l l l l l l l 2 2 2 2 2

Th

-37.,
UM

XVI

Fermentation Reactions

Source -- Swimming Pool

 

 

Culture' Type' Lactose ' SalicingT”Lannite T*lnulin ' hilk

' ' ' ' ' ' Acid' Clot
I T- I I I I I

201 'Alpha' + ' + ' + ' - ' + ' +
I I I I I I I

202 'nlpha' + t + ' - ' - ' +' +
I I I I I I I

2003 lillpha' ‘I' ' ‘I' l I- l + ' +' +
I I I I I I I

204 'Alpha' + I + ' .. ' .. I + . .
I I I I I I I

205 'Gamma' + ' - ' + ' - ' + ' +
I I I I l I I

206 'nlpha' + ' - ' - ' - ' + ' +
I I I I I I I

207 'nlpha' + ' + ' .. ' .. ' +' +
I I I I I I I

208 'alpha' + ' + ' - ' + ' +' +
I I I I I I I

209 Uglpha' + ' 4- ' + ' .. ' + ' ..
I I l I I I I

210 'Alpha' + ' + ' ~ ' - ' + ' +
I I I I I I I

211 'Alpha' ‘I' ' III ' I- ' III I 4" +
I I ‘ I I I I I

212 'Alpha' 1’ I - .' 1- ' .. ' 4.’ II-
I I I I I I

213 'alpha' 4- ' + ' .- ' .- ' +' +
I I I I I I I

214 'nlpha' + ' - ' _ ' - ' + I +
I I I I I I I

215 'Alpha' + ' + ' - ' + ' + ' +
I I I I I I I

216 'Gamma' + ' - ' - I - I + I +
I I - I I I I I

2]? 'Gamma' + ' - ' - I - I + I +
I I I I I I I

218 'Gamma' + ' + ' — ' + ' + ' +
I I I I I I I

219 'Gamma' + ' + ' - ' + ' + I +
I I I I I I I

220 'Alpha' + ' + ' - ' - I + I +
I I I I I I I

221 'ixlpha' + ' + ' - ' .- ' 4- ' 4.
I I I I I I I

222 'Alpha' + ' - ' + ' - ' + ' +
I I I I I I I

223 'nlpha' + ' + ' — ' _ ' + ' +
I I I I I I I

224 'Alpha' + ' + I , I _ I , u +
I I I I I I I

225 'Alpha' + ' + ' - ' - ' + ' +

II

III

IV

7

VIII

IX

Test
Substance

Eo hemolysis
Lactose

No hemolysis
Lactose
Salicin

Ho hemolysis
La 0 to se
Mamii te

No hemolysis
Lactose
Inulin

N o hemo lysi s
Lactose
Sa licin

No hemolysis
Lactose
Salicfll
Ilnilirl

No hemolysis
Lactose
1.1111111 t e
Inulin

hrnlolysis
Lactose
Salicin

Hmnolysi s
Lactose
lianni t e

Hemolysis
Lactose
Salicni
Inulin

TABLE i711
Eermentation Peactions

Source

Idouth
Feces
{:Jw imm i115

Loufii
Eeces
5 w i:- .n ‘1 in ,3

Louth
Feces
5 WI mm in g

houth
Feces

I 52711111111113

Lou th
races
Swimming

houth
Feces
Swi Ifll‘lil- i:

u

reces
Swdnmfl11~

Inouth
Feces
o‘ '-.'J immi n p;
Mouth
Eeces
Swhnming

1.5.0111; h
Feces
Swimm i113

Pool

Pool,

Pool
3001
Pool

Pool

Pool

Pool
Pool

Pool

lhndber
Reacii n3

. F’
UHJPJ CfiC)C CDNPO (NC)? «upcn <mcnm

OCfiCI

(DC;N> (bend cacn»

Str.

Str.

Str.non-hemolyticus 1

Str.

Str.

Str. h

salivarius

mitis

fecalis

pyogenes

emolyticus i

25

of no value in determining the source of the streptococci
from the pool water. According to Holman, Groups 17, VI,
VII, and XLare1meIely menbers of variations of the other six
groups, as he does not consider inulin as a valuable test
substance. duinit (25), in 1926, did some were along this
sane line using the classification and test subatances

as recommended by'Andrews and Herder. he came to the con-
clusion that the type predominate in feces is p33, fecalis,

whereas that which is predominate in the mouth is Str. mitis.

 

As in the work presented here he concluded.tkat the results
obtained were of little or no value, due to the fact that
both classes are found both in the mouth and in the feces
of normal individuals.

SUEMARY

Emwm.all the results Obtained, it would appear that
beyond a doubt the great majority of those streptococci found
in swimming pools have the nose and mouth as their saurce.
Following from this it would seem that the present use of
streptococci as a confirmatory test for intestinal pollution
is of little or no value. This would not mean, however, that
the test skmuld be given up, but rather that a different
interpretation should be placed upon it. It has been
demonstrated time and again that certain respiratory diseases
caused by pyogenic cocci may be transmitted through swimming
pool water. Although no hemolytic streptococci were found

in the pools examined, it was probably due in large measure

to some defect in the method of isolation. At the present
time there seems to be no practical method in use for con-
trolling these streptococci in swimming pools. liallmann,

in an investigation of the bacterial pollution of swimming
pools, has drawn up the following procedure and stain ards

for streptococci in swimming pools.

"The part played by the various strains of streptococci
in the respiratory diseases and their prevalence in the intest-
inal, buccal and nasal discharges maize the presence of strep-
tococci in bathing water very undesirable. Yet to eliminate
them from swimming pools would mean decidedly smaller bath-
ing loads and decided increases in chlorine residuals, either
or both of which would hamper the usefulness of the pool.

The committee calls attention to the fact that streptococci
tests are of value in passing on the condition of the
swimming pool water. A tentative procedure and stain ard
follows:

"1. Test for the presence of streptococci may be made
frcm the fermentation tubes used for the determination of
the colon-aerogenes group.

"2. Decent off the supernatant liquid in the fermenta-
tion tubes after 48 hours incubation at 5 °C. Smear the
sediment on glass slides, stain and examine for the presence
of streptococci.

"5. Standards. Not more than 5 out of 10 consecutive
samples taken on diiferent dates should be positive for
streptococci. The average streptococci index should no; be

greater than 2 per 100 c.c. of water for any considerable

period of t ime. "

27
CGECLUSIONS

Streptococci are not plentiful on the body surfaces
except where contamination from the mouth, nose and fecal
material has taken place.

Streptococci are found in.large numbers in the nose and
mouth and in fecal.material.

The streptococcus index roughly’parallels the colon
index only when the bodies and not the mouths of the
bathers have been.in contact with the water.

There is no relationship between the streptocoecus or
colon indices and the total bacterial count.

fermentation reactions are of no value in determining
the source of streptococci found in swimming pools.

Streptococci, as found in swimming pools, are not
indicators of the amount of intestinal pollution.

Streptococci indicate the amount of pollution which has
taken place from the noses aim.mouths of the bathers and are
therefore important in determining the safety of the water
in regard to transmission of sinus and reapiratory diseases.

It is therefore recmmnended that a streptococd. test

be made to measure the safety Of a P001 as far as respiratory

\

diseases are concerned.

\

\.

1.

2.

4.

5.

9.

10.

ll.

Llal lnlann, 27!. L.

Streptocaicus as an Indicator of Swimming Pool
Pollution

Am. Jour. Pub. Health, 18:771, 1928.

Manheimer, W. A.
Studies on the Sanitation of Swimming Pools
Jour. Infect. Dis., 15:159, 1914.

Grierson, A.M.M.

Observations on the dygienic Condition of Public
Swimming Baths

Jour. of Hygiene, 30:66, 1930.

Hasty, F. E.
Paranasal Sinus Infection and Swimming
Jour. amer. had. 58300., 89:507, 1927.

Prescott, 8.0. and Winslow, C.End
Elements of Jater Bacteriology, Fourth Edition.

Winslow, C.E.-A and Hunnewell, h.P.

Streptococci Characteristic of Sewage and Sewage
Polluted Haters.

Jour. ofjhed. Res. 3, H.S.502

Prescott, 3.0. and Baker, 8.x.

The Cultural Relations of Bacillus coli and
Houston's Sewage Streptococci and a method for the
Detection of These Organisms in Polluted Haters.
Jour. Infect. Dis. 1:193, 1904.

Savage, W.G. and Read, W.J.
Significance of Streptococci in Jater Supplies
Jour. ’yg. 15:334, 1916

Horwood, Gould and Shwachman

Indices of the Sanitary duality of Swimming Pool
Haters

Jour. of the Amer.Water Jerks Assoc., 25: 1933

Stafseth, H.J.

Mich. ngri. EXper. Sta. Tech..Bull., 49,pt 2, 1920.
Bryan, C. S.

Examination of.fiilk for Streptococci of mastitis
am. Jour. Pub. health, 22: £0. 7, 1932.

12.

14.

1'7.

19.

20.

21.

29

Kcrton, J. 11“. and Davis, G. E.

Bacteriostatic action of Dyes on Streptocazcus viridans
and Pneumonocci

Jour. Infect. Dis., 32:220, 1923

Llallnnnn, ”.a'. L. and Ca ~y, 21.

Study of’Bacteriological Methods of Testing and loans
of Disinfecting ".iater with. Chlorine

Am. Jour. Pub. Health, 23:35, 1933

Devereux, E.D. and LLallmann, W. L.

St: dies of the Technic to Evaluate the Efficiency of
Several Chlorine Sterilizers for Dairy Use

Jour. of Dairy Science, 17:351, 1934.

Smith and. Bream
Jour. lied. Res. 31:455, 1915

Bergey, D. H.
Differentiation of Cultures of Streptococci
Jour. Lied. Res., 27:67, 1912

Thro, W. C.

Experiments on the variability of the Fermentative
Reactions of Bacteria, Especially the Streptococci
Jour. Infect. Dis., 15:234, 1914

[‘1‘ "'

inrc, u. C.

Further BXperiments on the variability of the Permentative
Reaction of bacteria, ESpecially the Streptococci

Jour. Infect. Dis., 17:227, 1914

Holman, ‘.'i. L.

Relative Longevity of Different Streptococ ci and
Possible Errors in the Isolation and Differentiation
of Streptococ ci

Jour. Infect. Dis., 15:293, 1914

Gordon, Li. H.
Ready ”method of Differentia ting Streptococci
Lancet, 2:1400, 1905

Andrews, F.‘.‘.". and riorder, T.J.
A Study of the Streptococci Pathogenic for LLan
Lancet, 2:708, 1906

Holman, ‘J. L.
The Classification of Strepsococci
Jour. Lied. Res., 34:377, 1916

24.

Sdiottmuller, H.

Die artunterscheidung der Eur den menschen path05enen
Streptokohken durch blutager

hunch. med. Jchnschr., 50:849, 1903

{guinit’ R. I.

A Comparative Study of Streptocazci Isolated free the
College Swimming Pool, and from the Threat and Peces
of Healthy Individuals with Reference to Carbohydrate
Fermentation

Thesis for L.S. Degree

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