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THE SUSCEPTIBIIJTY 0F STREPTOCOCCI
T0 CHLO‘RJNE

THESIS FOR THE DEB-BEE CF 51. S
Mar-Vin F; Klang
1933

 

     
 

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THE SUSCSBZIBILITY OF Sfliifj 30031 90 CHLOYIIB
{A Thesis Submitted to the Faculty
of

Kichigan 3 ate College

agricultwre and Apglieu ocience

b1
{5

. TU”
flurv1n F. Klang
r the gegree of
Science

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TABLES OF CONTENTS

Introduction
Experimental
Pool Experiment
Laboratory Experiment
Technique
Tables
Discussion
Laboratory Experiment
Pool Experiment
Graphs
Conclusions

Literature Cited

8

l6

- 12

13

14

- 22

23

24

r1HE SUSCEPTIBILITY OF STREPTOCOCTI TO CHLOPINE

Introduction:

The popularity of swimming has increased enourmously in the
past decade; the result being the construction of a large number of
indoor and outdoor pools. Although methods of design and means of
disinfection have been greatly improved, methods of examination for
hygienic conditions have always been unsatisfactory. The methdds
of examination of drinking water have been used although the pollution
is not analogous to that of drinking water. In recent years the
American Public Health Association (1) method of water analysis,
with the name requirements for purity, has been used. Such a
Standard would appear to be very strict and judged from the quality
of drinking water, it should be a very satisfactory standard.

Escherichia coli is used as an idex organism in A.P.H.A.

 

procedure. This organism indicates fecal or intestinal pollution

W‘s-*m‘ -4 l

which in drinking water is important. Recently, there has been a

good deal of discussion on the reliability of the EEEEB.EE£1 index
when applied to swimming pool water. Some of the first work along
this line was done by Mallmann (2) in 1925 when he pointed out that

.Esch. coli is not a reliable index of swimming pool pollution, and

 

that streptococci indicate an unsafe condition. This brings up a
{group of questions: Are streptococci generally found in pools?

VVhat is the relationship between the amount of enteric diseases

grad. the respiratory diseases? Are the streptococci resistant to the

cannaunts of chlorine used in pool disinfection?

 

 

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contain ,

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the 00.13 pant (10113.;

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alonj~xith fo 3 from

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in ll~51:'Tal*"nn \

pollution f

in the pools.

SlLO pollution contained in the

*1
u 19

I" T“ 11089

5.;

-l would be very

~“ L‘ ~u4‘. '1‘“. ‘ . 'Pr w. '1. ”a -:
oi tnc mo ti and nasal yuSSm US tulle

would account for the

'reptococei were isolated from 30013

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001

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1 71- z ‘
.eeuntid hark lSOlwtOu beta strepococci

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from swim ing 90013 “n noston. rallnann an; tbe trite ii) isola ed
alpha and gamma streptoco Cl fire: :9 1' rithin the lent dcar.

The relationship bate ..-n Clit‘fl‘lc ii scams -ill respiutm'"
diseases has beta s?ugi*- by many workers. Tinnei”er (5) cites ex-
‘:)les OJ both of finesse types of disease“: as I‘C;1’W‘L'::1L b" the f0110~l‘”"
Te ce reports 3; chaos o: ente:ic lever ozon: the soldiers who
use; a STiHViLf 0001 “hi ch contain; so.-”e pollute ter, J
reported intestinal groteu31n11,0tion ‘fli?“ soldiers who bit ed
in the Dluuu iver, “”uhl attributes 49 Cases of typhoid fever to
bathing in the Clue, Chign re ortt3 415 cases of "acute““ frOm
bathing in a river, Tehr reports 3“ ca 0 of eve infection a egg

of a certain

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“1:112: pool, “chultz reports 18 Cases of

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at brown University, Omereba

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confrfictivlties ant clearinal otitis axons

'fllLT club. ‘erres hi self studied first Lam
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a.fwt;l else of meningitis cont: eted in a grinning pool. Hasty
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nnsel Sinus infe:tio;:. The “Kove re??? gee: Show that infections
of the eye, our, 1-39, and thr at j}: rally caused by pyaggnic

 

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Stregtococci noted.

Tie lvU retwrv me enst'ation of the susceptibility of stregto-
cocci was carried on using tlture of streptococci which were iso-
lited from swimming pools and haunn threats along with stock cul-
tures, one of which was the Decnez strein of scarlet fever. The
organisms were of all three types -- sighs, beta, and gamma. Ens
procedure of this 1sborstorj work follows:

Five one liter flasks containing 500 0.0. seen of college
tap water were sterilized by sntoclsving. (-eeweter was used in
order to approximate pool conditiOns.) One of the flasks was used
as a control and to each of the others Wes added a definite amount of
chlorine. The amounts 180d in this exp riment were 0.45, 0.52, 0.75,
and 0.90 p.p.m. Then a suSpension of the orgenisms was added in

uch quantity so as to give a count of 100,000 to 500,000 organisms
per c.c. in the flash. it definite time periods, nsnely, 15, 30, 45,
60, 90, 130, and 180 Seconds, transglnnts of l c.c. were made into

9 c.c. of veal proteose peptene broth (0.3fl dextrose). These later
were transferred serially to give dilutions of 1/100 and 1/1000.

These tubes were incubated for 24 hours at 37°C and the gr nth record-
ed. The number of organisms per c.c. in the flask was determined by

Inesns of the Helber counting cell. The results are Shown in tables

11 to 5. As a control, and check‘Esch, coli was used in this exyeriment_

111 plsce of streptococci.

 

 

 

 

 

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Techn 1 due :
L‘he streptococcus test was run along; wi th the regular water

The method consists of picking out

“ .
(“h-

ezzamimtions for :...,c-1, coli.

the suspected tubes and staining the rnterial. The suspicious tubes

were chosen by the presence of a vei. 13; growth which clings to the

sides of the tube and insert. Tibes showing this growth invariably

gave a positive test. The material was concentrated by removing

most of the liquor by suction. The sediment which remix-led was then

smeared on slides and stained. ri‘hese slides were then ez-unined

microscoluically for streptococci and the results recorded.

ri‘he Esch. coli is the test organism used in the A. P. H. A. test.

The pool water is planted in tubes ‘of lactose broth and also plated

in nutrient agar. At 24 hours the qumitity of gas was recorded and

a. plate count made. The tubes showing 10 percent gas were smeared on

eosin <- methylene-‘blue agar and incubated 48 hours. ..t the end of

48 hours the data were again recorded. All tubes now shoving; {:33

Typical Etch. col].

 

were smeared on eosin - metlnrleneéblue plates.
colonies are picks. from the eosin — methylene - blue plates and

planted in lactose broth to further confirm the presence of Tech. 0.2.11.

The same tubes are used for streptococci.
The sampling of the pools, as has been stated, is done with the

aid of the thiosulphate sample bottle; two smples being taken from

each pool. rThese Samples were taken at the same time but from Opposite

Sides of the pool. Samples were collected at times when the pool was

in use.
The residual chlorine content of the pools was determined by means

of n I-Iellige apparatus which used the colorimetric determination with

Go

 

 

 

 

 

ortho tolidine. The chlorine content of the pools ranged from
0.00 to 2.00 p.p.m. of chlorine. Since the pools used the same
source of water continually the pH effect upon th germicidal action
of the chlorine is omitted. A strict comparative basis was maintained
in all pools. The same method of detenuining chlorine residual was
used in the laborltory e periment. The source of chlorine for the
experiment was B. 3., a hypochlorite solution.

The factor of pool loads could not be controlled because the
pools used were in public schools and so the number in the pool
While the Sample was taken, was recorded. This factor is important
in determining the a cunt of chlorine needed to sterilize a pool.
The number of persons using these pools varied from 0 to 48 at the
time of saMpling. The effect of bathing load upon the chlorine residual
and the amount of pool pollution is shown in graphs 2 to 7.
"he culture used came from four sources, swimning pools, harm
throats, animals, and stock collections. The pool cultures were
alpha, and gamma streptococci; the lumen cultures were alpha and
'beta streptococci; the animal culture was a bovine genus streptococci;
and the stock cultures were alpha, beta, and Sauna streptococci.
(fie beta streptococci of this latter group was the Dochez scarlet

fev r strain. The pool cultures were isol ted by the writer.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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lee LuflOOTS, l to u, um useu 11 the cats sloc_s inuic,te the

.1 ...~ ‘ 1. - .40 4- ~- " 49, ' . . La. .. v‘ -
dilutior -n ”1121 fro -1 tool p11co. In this one these LVh"IS are
~' 1 L‘- .« . A? r? L’. f ° 1-,J. ° _ . ‘ ;.~,.. v 1-
coinci ent with tne lo; njr or o. ewe 1111t1ens tszd. Jhis LcthOd

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01 recoru1n; the iata has usee to av01u a more 1n.7e11dy svs 3m as used

in tile ori inzl recording of the duta.

The data iiom the lwboratory experimen show guito clearly th
range of s~1W:c 1t £1111‘3 of strcr1tococei to chlo;tine. One of the
““.n1)rs, a lUman 313 a streptococcus leing killed by the ta; water

itself, and the other exti e;e, a gangs stre tococeus isolated from
a swimminr poo l .‘hich grew in a 1/10 dilution after 90 second
exposure to 0.90 p.p.m. of chlorine. setween these extremes the
remaining stock, pool, human, and bovine cultures are arranged accord-
his to thL-i rdccroasin; resistance to chlorine. In 0.90 p.p.m.
availsble chlorine the two mo: t resistant orglnisms are a pool gamma
streptococcus and a stoe; sets strentvcoecus, t21e lat te‘ being the
Dochez scarlet fever streptococcus. Following these are a stock gamma

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streptococcus and an aleha str e11tococcus isolated from a pool. Since

the concentration of dilorine in swimming pools is generally nearer
0.15 p.p.m., an analysis of tie most res. stunt organisms of this group

follow.:s at this concentration. The four least stJoeotible organisms

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are in descending order - 2 pool gamma streptococci, a stoc: beta
streptococcus, anl a pool alpha streptcoccus. rhe susceptibility of
these orglni ms to chlorine is comparable since they grew out in
1/1000 and 1/100 dilution after three minutes exposure to 0.45 p.p.m.
of cthIine. The resistance of txese org nisrss to so 1‘91t a concen-

tration of chlorine is a Food arfnxment against the chances of pool

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sterilisation by lesser amounts of chlorine. Alth ugh board of
health pool reports previous to the use of the tiosulphute sample

bottle shes complete sterilitr in pools wherein the chlor1ne residual

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“'r n 1- .' O ' " I “‘ ‘ 0‘3 L. ' ‘ 1‘ - - a.

the less thin 0.~ p.p.m. “nu is the Sage ULMO the 1001s were over-
. ‘ .1 Q".J~1A 1 _ q ’ I . ‘J- ." 4. f . -~ ‘. '1 j -‘

losses .1tn eithers, Also Since ct--.t;c3c01 such es the Dochez

scarlet org-nism are 1uite resisten to rether ls 3e cunntities of

ab

chlorine, it 13 not ct all improbable that infectious disesses

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especially those of the rose ’etorw trict may be contrccted in pools.

Dhe data from the pool sunples were recorded and graphed according
to schools. The granhs she? the incidence of streptococci, fiseh. coli,

total count, chlorine residual, and pool loud by days for each

sample. The relationship between the presence of streptococci,
Vsch,.ggli, and total count and the chlorine residual is very marked.
‘ivery rise in residual chlorine being accompanied by a drop in the
amount of orgsnisms. This is to be expected, because of the germicidal
action of the chlorine.

The swimming pool data as graphs: ill*strcte very plainly the
following points: (a) the need of a streptococci index rather than
a hsch. 0011 index for standard swimming pool analysis, (b) the
greeter implied resistance to chlorine of streptococci as compared
with.Bsch. coli, (c) the relation between the chlorine residual
and.the streptocci, and 2222» g2;;_indices, and the total count,

(d) and the effect of pool loud upon.the pollution in the presence
of chlorine. Explanation of these point follows:

(a). The streptococci index is exce ded but once by the Esch.
cdli index in the entire experiment. fhis occurs in pool A (graph 2)

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uporl the fourth day. Thus, this would be the one day when.the A.P.H.A,

 

 

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nwthod'would be a true inde' of pollution, and the remainder

of the l-rs which show an excess streptococci pollution would be
unnoticed. The streptococci.yolhfiicn.would prob L13 not be shown
by the plates of the AWP.H.A. method since enrichment materials are

necessary to grow streptococci on solid sedia.

(b). Since the streptococci index is greater than the Bach. coli

index in all but one sanple, and since all of these sarnles are taken

from the same pools, it is implied that the streptococci are more
resistant to chlorine than Tech. celi.
(0) Graph 1 shows he relationship among the yer centum

quantities of 22g2, 392;” streptococci, and the total count. The
tadvantage of the streptococci as an index organism is shown by the
‘broad range of its growth in chlorinated pools. The streptococci
grow out in chlorine residuals of 0.0 to 2.0+ 13.13.13, while the
Iisch, ggli.grows out in chlorine residuals of 0.0 to 0.9 p.p.m. The
total count, according to the graph, coincides more nearly with the
Eggh, ggli.curve than with that of the streptococci, hovevcr, the
rise above the Eggh, ggli_curve may be due to the effect of the
streptococci count. This is highly improbable since the solid media
used was a plain 1utrient agar.

(d) The effect of the pool load upon pollution may be noticed
in many places on the graphs. The best illustration of this effect
is pool F (graph 7). The chlorine residual remains practically
(Huistant, yet when the pool load is high the streptococci pollution

increases. The chlorine content of this pool was at no time below

0.4.;p.p.m.

 

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23

Conclusions:

1.

The total count and_:ggh, gglirinde; does not parallel the
amount of pool pollution, as accurately as the streptococci
index.

Streptococci were isolated in pools containing 0.0 to 2.0+
p.p.m. chlorine residual.

Freshly isolated streptococci from the animal body were equally
as resistant to chlorine as those freshly isolated from pools.
as judged by laboratory experiments, disease producing
streptococci could survive in swimming pools for a period

of time sufficient to cause transmission of disease.

In laboratory enperiments there is no appreciable difference
in the susceptibility to chlorine of a pha, beta, and gamma
streptococci.

The use of streptococci as an °ndez of swimming pool pollution

is recommended.

'.-N'i

 

24

 

c 4-.- ~, .- r ', 1-" r 1‘ ---.-s"~ . ' ‘ - (‘ e"" -" ‘ ' . . "
stanlard Methods ior “naminntion of .ater and c a e- -7tl- E.itiOi

(‘

as all I:::Iic-..tor of wivimning'
lea lth 18:771, 1928

Falling-iii, '77. L. Streptococcus
I‘ool Pollution A. eO‘J'. i .b.

Ear..'ood, . P., Gould b. 8., and Shrachxan, H.
Indices of the Sanitary duality of
.;Lu~ Tool Vater
Jour. Am. Tater W rks Assoc. %5 124, 19?3

T'Tallmann, '3". L. nad 111mg, ‘1'. F.

The S‘uscet ihility of ?treptococci to Chlorine
with special Tlefsrez'co to Find 2:71: .-_in ' Pools
Eager presente? at the riichi' an acac new of Science at Ann Arbor 19-33

Iaiieirer, T. A. Studies on the Sanitation of Saim iing Pools
Jour. Infectious Diseases 15:159. 1914

Forbes, J. B. The hanjers of t
L“ cat 2

101 azichl brimming
"i. 1'. A. 89:507, 103’

It'Lllllitjlll, '.'.'. L. {4515. Gelpi, .1. G. 31‘.
Chlorine 3081717.: :;cc of Colon Batlli and
fitreutococci in a Gwimming _ ool
I‘ichi_j:..loi “11:. “‘23:. Qt. bulletin No. 27, 1970
hallmann, u. L. and Cary, 7m. Jr.
qtndy of Bacteriological Methods of Testing and
l'eans of Disinfectin; lather ".i th Chlorine.

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