5
4
0
§

'5:

4“, av
a
.
r
\

n A . ..
. _
..< .‘
. 1.x. ; y ‘ .
. v
\ I.
u
u h- I- } .
. ‘ .
.
n
. w, .
.
» _. .v: v - .
. u . 1“ .

..
‘ 3. 4

'Av‘

M. .. ,. .

~C. V.“
7 I
\

;.
‘-

vfi‘ L

3.0.".1. ..a 9.1. .vnlkril ,de‘tui.’ . u ...

n.
..
.g.
\.

I .1.
n,‘

...\._

A...

1

A Regort
on
The Viability
of
Suignin; Pool Or¢anisns
to
ficsidual Chlorine
a
T4-CSiS
SUUJittGd
to
the faculty
of the
Kichigan State Coliege
by
Lari w H. Lell
Candidate
for
Bacnelor of Lcicnce Degree

June 19:?

ReviLa 01 litu1: Lure.
ELuuctiOL of CLQaLiSLs WLLL [Kgose to Gil rine.
For gractical in“-oses 0.$-0.5‘;.).L. of availaale
chlL11-e is s fiiciunu 1o: 0H0 Lustructivn of 0195L1L43
illuicativ 01 100a; conta.i;at1LL in StiLLiLQ Jauls anon
engosLL 101 5 31L1LLL lLLQtL oi tiHUo finder 1Lu01z*

—

' 1': ‘- '1‘ '. ,‘\ 1 f! ' ~ x 1 'L "3 a . (v 1' (“ 1|“ 1 ‘ ' , ),",‘ ' t'\ I" ‘ ..~<‘ ‘ \
COLL1L1LLL u L hhbbfi 1L LLLLL J to ILLLVO Lll ~11 tLL

1.1.: _. ,.,. . _; .~. .,: 4.. "“1- .A'- . , | ,- _ ... .1. ._ ., w --.-~ (
ICLJLLLLLJ. 1-1114111“; o $4195.0le 111.11.511.11: 1...“ L Cu .L'w Lt L0
W! 't »r: 5‘“ ‘ "“"" 'ir‘ ‘ .z. . -.r-' €"-"' v-‘uv. " '. 4‘ '4 7 fl 1 1" "it 31W: " d“ ‘
1.111-L1 0...; Can‘toLJ-LLJ. A. “u LLVLLU c .L - Lamb“. .LlJ. Lair- 9404. ..L. (4.; u Duh. vI
\1"-~t- 1 . -,."'4.-----. a 1. " r-I». .TI".,—. ._ " ' ‘4 " ""
01 uliLk-L.*a’ CO‘-K..-L 0.5.01.3.) all“ l-Uk-Ubi \n‘..-tJJ~aI5 illcrfl‘ 18 a A.) gtvl-‘Jl—I—ltv
1.-" \ pl 1 1221* “’"J‘ 3‘ \‘I ’. ‘ "- "‘5" ‘L
b). (4.50341..;C “1-1.1.0.. UClhv JlLLLALLoo

In the 033; 01 a 31L,LL of tue 3:. mLin; 3001 water the

.. - . ' ... ' : 4.1-. ' u. ' J-r‘r-H . ..

reu1ct131 1s aroLLu 3Z,, LLo up or tLe reelsLLLL a1Lu11t¢

~~ -' r. I‘ v.
1‘..th t- {l.liLL 8.31‘ 004L194. S O

{‘3
*1
(‘3
F
L
H
p.

111Lr trec tLeLt with cLlL_1ne Let all B. CL 1 co L:LLL5
eggcar to no alige. 101 115*LLC0 Ln lit; s-lactouc age: the
00101-103 810 LucL LLaller aLd fail co ;r0u~CC aci . 1Lis is
cxdlaiLCd by the 1309 act tgc O1QCHLSQS a“c stunLCL Du LLe
action L1 th c-1011ne.

guru CL; turL- 511' ‘3'. cLli

. a, .: ~ ‘
0.13 C91. b31111."

g)

’C-I.

.1, -- J.
'L:1.e IJTLCL C.L1.L

laolc Cglur1ne to

H-

snomeg 1L8 rcuuctijn Vth 0.3 ¢.;.L. {vs
be LL.6’ a1 LLr tLL ii‘st 5 m1n t’s. inc iLitial comLts were
from 3,0LL to 500,000 in 1 0.0.. it is L‘iLt iLcL tLat LLL
initial rougctiOL anq not tnL JULcout roLuotiuu LLou;a go

CLLLiLerLQ as buC 901LLLt ILL Ctign

‘ "'1 "--: *- 7 :1 ‘:'-‘ :I-n
1:3:an LliL.LS 111 0119‘ 0.1:. 9.11-9.1. $1 JJUuElUIlo

Pa
*“5
C
(D
H
' ‘3
; ;
D
(I)
*7
F.

tits 5

1 1' \ - r. '- ~ _ 3' ' a ': 7.- v- "n n. .5 I _-~. ‘.~'\- . 4 ~ - ‘L " --1 4*

suitb out iuCu D5 Ub¢uu L o iuim*is (b tqe bbbb
O”' '(W ‘ .(N n2 .‘ 1‘0 .'r ‘ {N} x.‘."‘_ -- »,. ‘1 '4'; 4" ‘3 -.-'--. 1.- ....'...,.1 -
J-UCilélu)-.- ‘A-‘I-LL v-5.) ‘Du CL -4Ctl\L... - U &".)Cu-$-'.OJ- c-. [J6 J0 J; 95‘.) ‘a-Lfit‘m DU

COLBEC“ ggllauwo Lie lnltiCi CagLo UCLLQ egg/b.c.

P.J.H. cl. -crcczt lCuJCtiuH

2gb V
F\ ’ 1
uU ‘— '

- ' - x '3 - .. H ‘1 I ' ‘ ._. .7 “ -' F! ' -' 1. »' - v. ‘ ’ . "

(LCUCI‘CI' G. U. .-J€.C¢~..u’f,iu..o -—Jl-uo -UC‘J¢.'J v' 0 Lo \JO .' \zklb—I» \-/lu)
(”7‘ \ ' _f'v.' n.. .‘ -?. 1 ‘ (1., .. ‘ .-\ J.» n‘p‘,. h c‘

J.-'.L Lu vyo L2). Auk;4.--.(J . i.‘.b\4.LJ.u...g.

rh‘ . --. r-'1'\r".-' - "'“r.~. r, .. ‘5‘ «.-l -- \JW' .:““ r1- .2 " 'r‘
.Lux.‘ CO.-L.‘L5.LL7L1J'C Cum LILLLu DJ. 01.-.].041016 (u. 11“.» duh its:

ei'iicicz'xcy‘: :3 a CluOiLLLtLIL as shown 0;] all he LLCLiLD‘

(D
"J
C)
(‘3
P—
O
[M
p.
O
l-
C
H
u...
C‘
b
“3
C?
i-«
O
p
t
('3
0
c}
5..
1,.
0’3.
'1)
Fl
t1
(
'r

in its us

(3

Pb
f"

r“

}.J

l-

i

L ..
p...
y...-

L

C
H
C)
O

C uli'tl' :31
Inc sane Ibtort stresses tgo Lesa for tnc cagition of

ai x0083 w-CIe 0- Cnic matter is grc;cwt. igis is chesucru

to OVLICOJQ tge IUuiSLPLCU oiferca by the matte; an; in

ovechue the ozganisms 09401105 with thy materials tLic; woulu

0th rwise be leit “hibiicu The iact that 310? cr ""1Mms axe

n08115 as Largiul as a 1:35“ Lngucr neceswitatcu coggiwtc

td in. tu..0 38001. (LkU‘i'ulnt 01

C

-‘tiua; ii- oruur to insure sci

{:4
o
(”t
O.
Fl
0

A.P.L.f. Comgittuu on Chloxide if LiLC for gxim;igg gool

Dis OllieCtiUn. V01. 9).

Iothihg 00 1d we fOLLQ ii liters ure :3 to the results

I
I

592$

I

JCCU uh Strcgs. by the action of availtole cglorinc, in

tie nature cf gercentagc Ibufictiuns. Several auugugitics,
Loxncvcl‘, :5.;r‘e.i;.i.:f~.i‘.. 1:37.: ‘L 4b.}.-CJ- are .4036; Leaistmi to it; zctian
tugt {IU'LAJifiHBiOLEDrSo

The results given rugardiig the 5:5 iorneru are in
c ELQCtiGn with 31x)“ cultures, tiereiore it is IQ;OSLiJiu

to grxe an. cog33rlson of r aHLtS.

h

Purgose of the yroblem.

The ingortance oi fecal ochnisns in "Lllulr“ pools
has long been the interest of nan5 investigators and
esgecially tnose connected in any manner with tne groolens
offered by the QRHQCIS oi swinging pools

It as \;ith tnc desire to determine th‘ relative
resistrnce oi tne strains of Streptococci found in the peels
to those of B. coli and 3. aerogcncs tntt this proolom was
underts ken.

The two later organisns nave been used as a stand: rd
for a means of devising a method oi control via the cilorine
method and the gresence of Stregtococci has been no looted.

It is nitLout a douot this organise is as ingortant or even

m.

moreso than eitner B. coli or B. aerogencs.

Titn tnis fact in mine it lies been my desire to detergine
the relative resis ence of tiese organisms to eacn otier.

Leny thanks are due to Lr. ?.L.utllnin. and Ir. H. .Koch.

vno Le;e offered nan5 vzlus ole suggestions nuich have been

very beneiiciel to 4e.

Zliethods

After a thorough study of various netneds for the isolation
and identification of the organisms to be used tne follouin;
have been chosen.

For the isolation of B. coli and 3. eerogen—s a nethod
devised by Prescott and Winslow has been c.0sen, first because
of its singlicity and second because of its accuracy.

The method is as follows:

Place 10 c.c. of lactose broth in 100 c.c. of the meter
from which the organisms are to be isolated.

Incubate at 37°C. for s4 hours.

Place 10 c.c. of the above incubated seuyle in each of (b)
fermentation tubes of GOLch strength lactose broth and l c.c.
in a tube of single strength.

Incubate these tubes at 3700. for lB-Ld hours or until gas
aggesrs.

From the tubes showing ’88, streak Lg eosin-nethvlene-blue
a)

take duplic:tes for 20° and 37°C. incuoations 3rd incubate
for 24 and 48 nours.

Pick tygical 3. coli colonies union are black with a
metallic sheen and B. aerogenes which are black nitncut the
metallic color and confiru tith the Vogues-Prosenauer and the
Methyl-Red tests.

The V-P test is undo as follows:

Pick the typical colonies and plant in dextrose broth.

Incubate for 5 dsys at 37°C.

At tne end oi the incubation Jeriod {dd s c.c. of a low
solution of potassium hydroxide (KCL) to o c.c. oi tge u day
broth culture.

Hold this solution over niuht ior tne iorustish of the
eosin-pink color, which constitutes a positive test for tne
B. eersgenes.

The Iethyl Red test is made as follows:

Inoculate the dextrose tubes Witfl tggicsl_colonies and
incubate for 5 dais.

To 5 c.c. of the culture add 5 dress of nethgl red sul-
ution.

After a snort period of incuustion a red or Veglov color

c4

will develoo. Ehe red is JOSitiVC for tne gresencc of B. celi.
“he Vogues' rosehauer and tethgl-Red scnene is:
B. coli B. ceroqenes
V-P - V-n /
23-11 / 1:-3 -
The broth used in the tethgl-ned test has a number of
sgocisl invredients snerezs tnst used for tne‘V-P test is
plain dextrose broth. lhe iorner is nade :8 follows:

Distilled neter----—----------------------lOUU mils

Di-gotassiun-hgdrogen ghosghate----------- 5 gross

Dissolve the zinture of rsterials over a iree flags;
make u} to volume. filter and sterilize is: so minutes at

ls :.0‘;=.1;d- pressure.

‘

C]

The Ietth-Red indicator is nzde b; eis-o;vin¢ t.l grass

of methyl red in 300 c.c. of achhol and diluted to Set c.c.

\1Ltil diffitillCd TICLCIIO

To isolate StICJtJCOCUUS the ester sangles used to
isolate 3. coli and 3. aerogcncs {re germitted to incuuate ior
Eé-td “ours or u til a sedinent occurs 'n the tuoe. The

su ernatax.t iluid is rczzoveu anu t e seei-.1c;.t s ‘si:.ed and

enanined microscogicelly for the gresence of the organisns.

J
I

If tned are gresent : snail portion of the seeinent is sfis-

I

pended in saline and tnen Sg~nred on 1.5, liv r (QCI slates.
The colonies are visible OLlJ after t-b dads incl cti.n at ;7UC.
and are very unite and usuvlly gin Joint in size. Ihe isolated
colonies are ensuineu again hierosco icelly ior their true
identity, if gssitive liver auzr slants are strecled.

As the virbiLitJ'oi the Stregs. is more or less linited

it has been iound neces er; that transiers of tnc culture be

made once in do hours.

To determine the amount of available chlorine in the test
solution a.uethod devised b; E. B. Buchanan and R. G. Perkins
has been englo;ed nitn satisfacti n. The method is very single
as well as rapid therefore making it esoeciaily convenient for
a test of this nature.

Two 2' x 0“ blocks 15" in length are fastened together on
the flat surfaces and holes sufiieiently lrrge to permit the
insertion of lestlers tubes are made throu¢h the six inch
dinension. Fourteen oi these tubes are placed in the block and
numbered and t 0 following amounts of reagents are glsced in
each tube:

Cl/p.p.m. CuSOU/c.c. KLCr§07
0.01 0.0 0.8

0.0L 0.0 2.1
0.03 0.0 57b
0.04 0.0 4..)
0.05 0.4

0

0.06
0.07 1... 7.5
0.03 1.0 0.7
0.09 1.. ~.;-.0
0.10 in. 10.0
0.20 1.9 . 00.0
0.;0 1.9 ' 30.0
0.00 2-,.0 88.0

0.50 200 45.0

A ruoser stooger is fitted tit; a iasoeroen tuoe rue the
closed end of the tuoe ,ergitted to hrojeet tgrouQe the seell
end of the stopoer. this permits t¢~ closed end of t-e tube to
be iflgchCd in the standard. The reek is so constructed th:t
light is perfiitted to ass throggh the bottou OI tee tuoe,
therefore ween lOOkiL5 tnroupn the l Le 01 t.e stagger tLroth
tie small tube it is gossiele to make a comgerison wits greet

ragidity and a degree of eeeurae; ootainaole onl; by geens of

a colorimeter.

He 011od for t1 0 test.

A small {meant of chloride of line is adaee to about
three liters of sterile ta, water are tle mixture stirred
well and Ier‘itted to stzld ior at least thiltJ minutes.

A tseLt -1o 1r 0 .lture oi the orUanisJ to be tested is
wasaed fro; the liver aUar slaL. with sterile Lfllihc. A 1003
traLsf r is made to a iarU r quahtit; or saline. Ihe number
of orU11e13 in this susgeLsion is deterLiLed bu acres 01 a
heemccgt1.ett 1. If the Lamoer desires in diiierent than that
in th s su91eL910L either diluti01;11t- saliLe or adtitien
of the culture may 00 hzde. “Oh the nauser 0e; ired is 00to1ned
in the SQSJGhGioh four (Latoo rre Lace to check the actual

number. 1wo plates of the oriUiLal BuSgthlun are -1ade by
using i 0.0. agoahts aLe two of a 1/100 dilution usihU é c.c.
311-0111. L s .

At a convenient tine a portiOL of the oriUiLal susgeLsion
is adaee to the sole tioL coLtaiLiLU the ChloriLe, the aaouut
of residual chlorine having been determined and at the end of
l, 3, 5, 10, 15 and 30 LiiutciLterv:is L c.c. aaouLts oi the

test solutioi is reaeveaane ihhediately plated. The plate

beiLU poured Lith the liver aUar.

It is in errtive thrt the glate be ,0urcd iauediatel;
after the portion hrs been rehevee to a"rest the action of

the chlorine seiLU reLGV0d flit; tAC irggie, also it is desir-
able to hive the tCJ, rcture oi the (Uzr suiiicieLtly low
that it till not hare tle orUaaisLs as their resists Lee to

heat would be lowered due to the (etioL of the chlorize.

The plates are inverted aiter cooliLU end iLcuoatcd ior
ttenty-four hours at 07°C. aiter which tiLe theJ are couLteo.
"ed ior the

The countiLg giate of Zolilugel's 0e1LU 0331

Q
.3“:

purgose. (Giltrer's Lao. fianuzl PaUe be)

Fr u the noaber of colonies UremiLU the gCICCLt oi the

gran. ...‘ m c .— (I - ‘—. . «‘4- - ...-.‘, :
orua1ishs destr gee 1s 000e111heo.

t.)

Results.
TLe results of a test usiLU 3. col_ as the eLgcrigcntel
orUenism is as follows:

A -.-.-'-»~, . . . ,
fibutht .o 9.0.”.

" " ~-2~ '7' '1‘ 0 ~ N- -'\ -r- .4 ...\,".- 4’- . r
1110 01 ex; s1re Goes re1ceht isolation

l m” 1 Ar- (szfi
Lao-0"; ’ - J V v . _ ‘4
P" ._.. - ‘. 7 I. '12: .‘J I! ‘
U .JnLLl 1,-L‘au va‘ovv

5 min L4! 90.6.

1 sin :.509 0e.00
0.min @9114 34.10
5 min 1,810
10 min 1.434 93.45

15 min 094 93.63

C:
C
E
H.
s
(23
35-
.q
('3
( ‘I
C
(D
K}

- r .
obi) JIQILJOLA-l.

.020 97.74

95.01
90.2;
96.90
99.17

"J‘J 0 LL)

..
l

13.;11

1.1.1
C)

[-4

u min

o... 1. 1:11 I].

1 Lin

9. 171.1 1').

lo his

to min

1 min

0 ;in
1v 3's

_ ‘fifl'; 2'.
1" 10“.“

L. J Lilli

.07 ;JOQOA—l.

U. ‘lUU

,.
~ 5).

‘1 .U {It

s.ve;

L/U " - _.-
I I J.
o 1 0* o A.

"1- ‘ . '_
.‘Jz *Jo‘.o-.10

U. .. 1.)“)
5“
K .L/‘w'

'-
l. O

O, 0-,. (1
‘u’I-JKJJ

c,
C.)

O
) t! f.
H H

98.00

Ihe oriUiLrl susgeLsioi 00 st was 100,000 erUenishs

A.

:er cuoie centimeter. The teaserature

_ ' n' (--O
‘\.C' D.) ‘0“ C .

The results oi a test usiLU J. aeroUen

J

&

orUanish is as is 7 ts:
AUainst

frise oi LLJOQQTC

l 31L

0 11111

0 min

10 min

it min

u b 1.111";

1 min
- min
0.min
10 min
lo min

00 min

1 min
0 min
5 min
10 min
10 min

LU min

'? ‘

00.1111}

6‘.

t“ 1

.14 ‘.’o;)oi.lo

H 1:3
3 C

.09 3.9.m.
CV40
~70
Lee
106

47

(1 " “" ['0
L; a...) bi-» tent

percent reduction
99.91
00.01
93.99
93.:
103.00

leboOv

2/39. 9;-
.39. 91"..)-
-9 00

.l/V

0.7)

[A

v . 0 b v

. r .‘

9 \3 ._ .
t . V V

100.00

01.0.;

$21.99

(JV

2111;
Liill
min

13111

organisgs

:C

J;

"I

.L

.04

$ A.

’1 1.‘ ‘
UUIJ

55

175

V“

P'.‘

cuoic CULLiQVLGI.

tuggcratglc gave.

8

O

0)

3.3.11.

11;;

1.0.41. t: L)

SUSQCLSth COhtLlLCu u..,.

'11.).

m,-

r
C.

'4

1"
a

«39.57

0...} . 1."
u’ v . LJ J

r‘ 1" f)'
. 'n \ |
tJd. db

,. xi»
avovd

O U“.

P].

g V

‘;;e rssults o; a tss. using Stregtococcus as th; t;st

C)
5’
H
H
C"
O O

‘\ , I q‘~~ '7 . ’ "
AbillLfSt 0t) 90;).-.10

Tigc of 6:; sure C unt fiercest reduction

1 - '0 —- ~ a " ‘ r‘ "r,
X: 13.1.11 11).." , 700 ls) o bl
‘1 , .' . ,9 f') t .‘ 4:: (\
‘__" I.-.Lll. ‘;.Q7J ()5)’ V

i min 23,;Jé “O.Ls

'/ {To 9' l ‘
(-9 a.llL 150.59 I U J4: UL;

u 7 (1 ‘ {I
I.) l L..-lt b I "A-H’ b 0 v

.- 3. .1 ‘a
iv 1.114.; . Hui“: Kv‘ 70 («l

P
C
ya
i
i..-
t
L
C; ,
C‘
C
I
O
C":
L; .

.1 9.3.3.
+ min L00,CUU li,1d
7,- “gm ()4 7:0 rm “L.-
.. '11 9.... Q Md,‘

--l, '“ . UU’ DUI}; U".(‘")

a min 12,215 -L.La

H
C
L.
t“
C -.
C
x}
(V
C:

L-
C
O

( I
H

1. 111.11;

‘4 fi-A‘.‘
0 Lin
10 min

H
C

‘ <
z.)
|-J.
H
0—"

1 mm
9' L11;

4-.

U ;.l 1:.

la mih

Qu min

.07 p.

.04 p.

A.

p.m.
LC,7GC
1;,Scz
17,2)fé
l:,s-d

ls,¢$4

4,;14

p.u.

O .

21,64”
1s,;;2
14,764

5,ssu

£30.41. ’

58,-z0
r. ; , L71";
------
Lé,&gz
LCfiulG

Ls,400

90.77
91.77
92.01
bé.ll
9b.¢0

96.13

90.22

87.12
s7.54
-----
£9.96
6;.s;

95.91

The original tea» suspension contaiscg LL5,0UO organ-

isss ger cuuic ccst446ter. The test vas run at

iirst two tests included platlngs at i tsrvais of loss t”:

one minute to gain sose idea as to .sc rate of destruction

suriLb tse first minute.

dis”

Conclusions.

:48 CKfiCriMULtCl results of tgis test a”c in keuoisg
with tsosc found is the : Vic: of litoxzture. It is ksoxn
to 30st :srkors tLat tso Lostructioiof A. co-i rad 3. scr-
ogsxss is nearly loo gorccst is QULMtiQHS COhtlixldu .s .g.s.
sum over of availaoio calorino and that any focal strain
Ex: 0 12.1.8]:

or Stro,tococcus is mars rcsistsz' than sit er oi

o organisxo rcnaisin; are

~J
O
H
O
f x
("

organiszs tasted. The 1
call;& th Insistunt nisority {so rrs or solo oi Litmstanding
.4 3.3.;. for 8.0“t iuLQLVfilS oi tige.

It is to so oxgoctoi to find stiegs moro r sistsut to
the efiocts oi chloxiho discsioction as it is sits. gussioie
in iii-nu instances 1;.) i‘i.d t 0;; growl-5t in tse amiss... of
fermentation tuoos that show negative results to gas iornsrs

0.5. J. 0011 and B. aorogcucs.

 

 

"“1

THESIS 1m. HALL 3.3
REPORT ON VIABILITY 0F SWIHMING
L POOL 0RGANIS"S T0 RESIDUAL
» CHIDRINE 1927 MPH

 

. ROOM -
BORROWER 5 NAME NUMBER

 

MICROBIOLOGY A
PUBUC HEALTHND
LIBRARY

 

 

"'iflifimsiflflflifligfinfififlfljljlilimjm‘tilfifiifim“