III

I

I

II"

II
I

I
I

I

III
II

III

III

II
I

I

III

I

I

I

I
I

 

._{
13x3 1|
.CDLOCfi I

A STUDY OF DIFFERENT MEDIA
FOR THE DETECTION OF MINIMAL
NUMBERS OF STREPTOCOCCI
IN SWIMMING POOL WATER

The-sis for the Degree of M. S.
MICHIGAN STATE COLLEGE

WCIIS A. ShuIIs
1939

: . IIEEEEE. .
. . 5 Ara...&...,......£.¥.,...

 

-fil , 0.
a... it _ . . .
.1 Y . _ I .‘ . . I . . r W»

‘

 

 

‘A'

t

.V’

‘0-

 

o‘v '

 

 

Michigan State College
of

Agriculture and Applied Science

A Study of Different Media For the
Detection of Minimal Numbers of Streptococci

in Swimming Pool Water

A Thesis
Submitted to the Graduate Faculty

For the Master of Science Degree

Department of Bacteriology and Hygiene

by

Wells A. Sgulls

East Lansing, Michigan

1939

THESIS

 

 

 

 

Contents

Acknowledgement
. Introduction
Historical
Experimental

Procedure
Discussion
Conclusions

Literature cited

Page

11

12

16

18

Acknowledgement

The writer wishes to express his gratitude to
Dr. W. L. Mallmann under whose direction and guidance
this work was conducted and to other members of the
department of Bacteriology and Hygiene for their help-
ful suggestions. He is also indebted to the Lansing

Health Department for its cooperation.

Introduction

A study in this laboratory by Darby and Mallmann (l)
paralleling this presentation, demonstrated that more coli-
form organisms would develOp from minimal plantings with a
tryptose lactose medium than would with lactose peptone
broth made according to specifications of the American Public

Health Association "Standard Methods of Water Analysis" (2).

This study was initiated on the thesis that this same
medium or perhaps a modificati n would allow the development
of a greater number of streptococci upon minimal plantings

in a manner comparable to that evidenced with the coliform

organisms as cited by Darby and Mallmann.

Although the basic findings of this study are more or
less applicable to the cultivation of streptococci from any
source, the studies presented are applied primarily to the
isolation of streptococci from swimming pool waters. The use
of pool water as a source was selected for three reasons,

(1) that swimming pool waters contain both Escherichia coli

 

and streptococci introduced by the bathers, (2) that samples

could be obtained with ease, and (3) the degree of pollution

of the swimming pool could be controlled.

The importance of streptococci has been shown by

Mallmann (3) who found that the incidence of streptococci

-2-

paralleled the amount of pollution more closely than did

the Esch. coli index.

Studying the pollution indices of natural bathing
places Mallmann and Sypien (4) found that the streptococcus
index fluctuated with the bathing load and that streptococci
were not found at points free from bathing pollution. Their

findings were also true in indoor swimming pools.

The importance of streptococci in swimming water is
further demonstrated by the fact that these organisms repre-
sent, contrary to most Opinion, a measurement of respiratory
contamination. In studies by Mallmann and Ardrey (5) it was
shown that the streptococci found in pool water were of res-

piratory origin.

If Esch. coli were more resistant to disinfection than
other organisms encountered in swimming pools the present
test would be satisfactory, but Klang (6) has shown this is
not true. It appears that the streptococci are the more resist-

ant to the chlorine in the pool and therefore the more important,

particularly in the light of their sanitary significance.

The use of various media was studied by Habermann (7)
with the result that lactose peptone broth was found to be the
best of all the media tested for the growth of streptococci. As
the streptococci present in a swimming pool should not be in

great numbers, a medium functioning well for minimal plantings

_3_

is needed. Thus the medium containing tryptose was used in
the hope that such a medium would show higher indices than
lactose broth. With these ideas in mind the following experi-

ments were conducted.

Historical
One of the first mediums mentioned for growing strep-
tococci from water supplies as a routine method of analysis
was made by Prescott and Baker (8). They planted the water
sample in lactose, dextrose, sucrose, and other sugar broths
and on litmus agar. The streptococci and EEER-.22£L were dif-

ferentiated in the litmus agar by the difference in reaction.

Continuing along this line of color differentation of
the streptococci, Todd (9) found that with neutral red in a
one per cent solution in lactose agar the streptococci gave a
typical color reaction. Several Species of streptococci could

be distinguished by this method.

Crowe (10) using blood for enrichment prepared a medium
using Hiss serum water to which he added 10 per cent of a
boiled blood filtrate. This was prepared by thoroughly boiling
one part of defibrinated blood with three parts of water and
autoclaving the filtrate. In 1923 Crowe (11) used a special
glucose-blood-agar medium which grew streptococci well and upon

which they showed different morphological charactistios.

Using a medium which contained meat infusion, peptone,

_n-

gelatine, casein, glucose, disodium hydrogen phosphate, and
sodium citrate. Ayers and Johnson (12) grew streptococci well
and kept them alive for over four months. The reasons they

gave for this growth are: " consistency of medium, presence of

casein, slight amount of sugar which serves as an easily avail-
able source of carbon, and the favorable buffering of the med-

ium.“

Crowe (13) used his special blood agar to differentiate
various species of streptococci by the color of the colonies.
Continuing with the principle of blood agar, Spray (14) used
an agar containing 5 to 10 per cent of a fluid, which he obtain—
ed from fresh blood clot that had been digested. This medium

grew streptococci abundantly.

Using a culture of streptococci grown in blood agar and
a culture kept alive by passage through rabbits, Beckwith and

Rose (15) found that (l) the passage strain had a more active

proliferation than the stock culture, (2) that the passage

strain was more virulent, and (3) the passage strain had a

lower thermal death point.

Medalia, Bailey, and Atwood (16) developed a modified
Loeffler's blood serum medium which they used in routine examin-
ation for streptococci. This medium called for pig serum pH 6.8

which after autoclaving gave a solid transparent medium.

The strangest medium developed is that of James' (17)

-5...

using a 20 per cent infusion of Quahaug (common American
round clam) or five per cent of fish roe infusion. The infus-
ion seems to supply the necessary growth substances. The
growth on fish roe equaled blood infusion agar, but a decrease
in growth took place if the concentration of the infusion ex-
ceeded five per cent. The medium consisted of the infusion,
Witte's peptone, sodium chloride, agar, and 0.5 m1. of sterile
defibrinated sheep blood which was added after autoclaving and

cooling to 45°C.

Wright (18) found that the size of the streptococci
colonies is influenced by the thickness of the layer and the
concentration of the agar, the addition of blood or serum and

the availability of the foodstuff.

The size of the colony is not affected by omitting glucose
from the medium if peptone is present in adequate amounts.
Filtration through thick filters may cause marked deterioration
of the broth, and it is best filtered through glass wool or thin

paper.

Going back to the earlier work on the selectivity of dyes,
Chapman, Lieb, Berens, and Curcio (l9)produced a medium which
contained bromthymol blue in lactose agar at a pH of 8.6. With
this medium it was possible to differentiate by color differences

pathogenic from non-pathogenic streptococci on initial planting.

In order to grow Strgptococous salivarius, Saffor,Sherman,

- 6 _

and Hodge (20) used a medium made of 1.0 per cent lactose,
0.1 per cent glucose, 0.3 per cent meat extract, and 0.5
percent each of yeast extract and peptone. Sherman and Stark
(21) found that gas could be produced in lactose if
fifigeptococcus thermophiligg (lactose positive) and Proteus

(lactose negative) were grown together.

The rate of fermentation of Streptococcus lgctig was
studied by Rahn, Hegarty, and Devel (22) using a 2 per cent
phosphate buffer‘%-2 per cent glucose # 0.3 to 0.5 per cent
peptone. The Optimum pH was found to be 7.0. The most rapid

rate was in a buffer solution containing h per cent phosphate.

In considering this subject it was thought advisable
to make a very short survey of the nutritional requirements
and the biochemical reactions of the streptococci. Lloyd (23)
found that certain accessory factors of growth were present
in blood, serum, milk, and animal tissues and vegetable tissue,
they were soluble in water and alcohol, but that they could
be absorbed from the medium by filter paper. He also prepared

a tranSparent blood agar.

Later Cole and Lloyd (24) concluded that the important
factors necessary for growth are a suitable pH concentration,
a high concentration of free amino-acids, and the presence of

certain special "growth" hormones.

In the study of streptococci metabolism, Kendall, Day,

_7...

Walker, and Ryan (25) concluded that carbon was the source
of energy and that it was indispensable for the structural
requirements of the organisms. Ayers and Mudge (26) shed
some light on the composition of the "growth" promoting
factors of Lloyd in vitamines. They used an autolyzed yeast
extract containing these growth promoting substances. The
water soluble B did not appear to be of significant value.
Fats and oils, vegetable, animal, and mineral even in very
small amounts were found to stimulate the growth. They con-
cluded: "either the growth promoting prOperty of fats and
oils is not due to fat-soluble H, or this vitamin is present
in mineral oils, or the stimulation is due to different
causes in the case of the vitamin-containing fats and oils

and the mineral oils.”

Whitehead (27) found that on tryptic digestion of
caseinogen the digested mixture may be separated into three
fractions with Ethyl alcohol. Thus he was able to remove
all the inorganic phosphate present in the fractions by
precipitation with 66 percent alcohol. There was a decided
lag in media prepared from the two fractions containing no
inorganic phosphate.But if phosphate is added rapid growth
occurs. He concluded from this that at least three fractions
appear to be concerned in the growth of streptococci; two
are probably protein derivatives and the third is phosphate
preferably in an inorganic form. It appears that the phos-

phate catalyzes the reaction.

- g -

Hucker (28) found that the strains of streptococci

producing levo-lactic acid hydrolyze lactose faster than

other strains.

Krashaw and Gies (29) developed a liver-peptone
medium which was useful in storing streptococci. They were
successful in keeping cultures three years on this medium.
Considering the amino-acid requirements of streptococci,
Hutner (30) used deproteinized skimmed milk and a casein

hydrolysate demonstrating these requirements.

No historical review of work on media would be complete
without some short resume of the literature on the important
subject of hydrogen-ion concentration. It is a known fact

that the hydrogen-ion concentration plays a big role in the

success of a medium to grow bacteria.

Using electrical pH measurements, Ayers (31) found that
the final pH for streptococci from humans was 4.6 to #.8 from

broth that was pH 6.8 to 7.0 initially.

Grace and Highberger (32) found that a broth with an
initial reaction of pH 6.8 will favor a more rapid growth

while a broth of pH 7.6 will distinctly retard growth.

The final pH of glucose or lactose broth produced by
streptococci was found by Foster (33) not to be influenced
by the presence of KgHPOh in concentrations up to one per

cent providing sufficient glucose is present. He believed

-9.—

the optimum pH for acid formation to be 7.8. He concluded
that acidity is the chief factor in inhibition and death
of streptococci. A table for minimum and maximum growth

is also given.

In studying the variation in pH of broth media
Foster and Randall (3n) found that there was a change in
broth of 0.2 pH upon autoclaving. In general the pH change
was in the direction of increased acidity (decrease of pH);
thus the greatestpH change would come in broth alkaline in

reaction, less in neutral broth, and the least in acid broth.

Experimental
The supply of water used in the following experiments
was obtained from five secondary school swimming pools and
one out-door swimming pool. In most cases these pools were over
loaded. Of course there were times when the pool load decreas-

ed to only a few bathers.

The samples were collected in sterile thiosulphate
bottles, as recommended by Mallmann and Cary (35), and taken

immediately to the laboratory and tested.

The lactose peptone broth and nutrient agar uSed in
the eXperiments were made according to standard methods of
A.P.H.A. (2) the tryptose broth and agar were made as follows:
2 percent tryptose, 0.5 per cent lactose, 0.4 per cent KEHPOh,

0.15 per cent KH2P04, and 0.5 per cent sodium chloride per

_10-

liter. The agar contained 2 per cent tryptose, 0.1 per cent

dextrose, 0.5 per cent sodium chloride, and 2 per cent agar.

In order to maintain the concentration of lactose and
peptone as recommended by A.P.H.A. "Standard Methods of Water
Analysis," (2) the broths were made with twice the necessary
concentration of peptone and lactose; so when a 10 ml. sample
of water was added to 10 ml. of broth the concentration of
the required 0.3 per cent of peptone and lactose was reached.
In order to determine the smallestamount of sample to give
positive results various sized samples of water were used in
the 10 ml. of broth. All the broth was tubed, 10 ml. per tube,
with a Durham's gas insert, adjusted to pH 6.8 and sterilized

at 15 pounds pressure for 20 minutes.

The culture used for comparative tests on the media was
a non-hemolytic streptococcus isolated from an ulcer in the
colon of a man. This organism was carried on 2 per cent tryp-
tose and 2 per cent agar slants. A hemolytic streptococcus
isolated from a dog's kidneys was tried, but it grew too scant-

ily.

The broth used in the growth curves was the same as
above, 100 ml. of single strength broth to a flask. All incu-

bations were conducted at 37°C.

A short resume of the procedure follows. At least four

samples were obtained from each pool for each series of tests.

..11..

The samples were collected in sterilized thiosulphate
bottles as aseptically as possible. The samples were
transported to the laboratory immediately. Five 10 ml.
portions of each sample were planted in 5 tubes containing
10 ml. of double strength tryptose lactose broth and 5
tubes of double strength lactose peptone broth. One ml.
portions of the water were plated in duplicate in standard

dextrose agar. The tubes and plates were incubated at 37°C.

with a 2M hour and a #8 hour observation. Any gas was
noted, a loopful of the broth showing gas formation was
planted in 10 ml. of brilliant green lactose bile broth for

confirmation of coliform organisms.

After the 48 hour observation Gram stains were pre-
pared and examined for streptococci. The streptococci index

was determined in the same manner as the Phelps colon index.

The procedure for the growth curve was carried out as
follows. An appr0priate amount of the organisms was scraped
from an agar slant with a 100p and put into a 10 ml. saline
blank to give a faint turbidity. This was then diluted to
1:1000 and one ml. portions were seeded in 100 ml. flasks of
lactose peptone broth and in 100 ml. flasks of tryptose
lactose broth. The flasks were shaken well and 1 ml. was
plated in tryptose agar. The plates and flasks were incubat-

ed at 37°C. At three hour intervals for twelve hours suitable

dilutions of the broths were plated in tryptose lactose agar.

-12-

The plates were incubated at 37 C. for 43 hours and counted.

Duplicate plates were made of all dilutions.

Discussion
As stated previously this problem was undertaken to see
if tryptose lactose medium could be used for the isolation of
streptococci from swimming pools. It is true that lactose
peptone broth does grow streptococci from swimming pool waters,
but it is questionable whether or not this medium allows the

deve10pment of all of the streptococci present.

Portions of five 5 ml., five 1 ml., five 0.5 ml.of
water were used in 10 ml. tubes of tryptose lactose and
lactose peptone double strength broth respectively, in order
to determine if these amounts would show any variation in the
number of positive tubes. Five 10 m1. tubes of the broth
were used as this number was easy to handle and was large
enough to reduce most of the errors that might be introduced,

In the above series of tests it will be noted that
double strength broth was used rather than single strength
which is recommended by standard procedure of A.P.H.A. These
concentrations were used purposely to determine the tolerance
of the streptococci to varying amounts of tryptose, peptone,
and salt. Double strength tryptose lactose medium contains
a high concentration of tryptose (4 per cent) while the lact-
ose peptone broth contains only 1 per cent peptone. With the

portions used there would be very little if any dilution of

the medium. The results of these series are very interesting.

-13..

There were only five samples tested using five 5 ml.
portions. Samples tested in the tryptose lactose broth have
a higher streptococci index than those tested in the lactose

peptone medium. The mean Phelps index is 17.6 for tryptose

lactose broth and 14.4 for lactose peptone medium.

Fifty four samples were tested using 1 ml. dilution
series. Table 1 shows that the streptococci index is approx-

imately equal for the two broths. The mean Phelps index is

the same for each broth, namely 63.4.

It is evident from the above, that with the Samples
used the dilutions tested were not high enough. Too many
streptococci were present in each tube, so that sufficient
organisms were present to allow growth in both lactose peptone
and tryptose lactose broth media. With the 0.5 ml. portions
the dilution was such that the tubes received minimal numbers
of organisms. 0f the 22 samples, table 2, used in this series
the tryptose lactose medium index was higher than lactose
peptone broth index. The mean Phelps index was 17.12 for
tryptose lactose broth, and 16.86 for the lactose peptone

broth.

These results can be shown graphically by plotting
thefrequency of the number of times the Phelps index of the
sample occurs. Graph 1 shows the 5 ml. portions and the 0.5
ml. portions. 0.5 ml. portions contained, as stated before,

minimal numbers of streptococci, this curve shows a tendency

'TABLE I

A comparison of standard lactose peptone broth
and tryptose lactose broth using 5 ml.

          
    

       
  

  
  

   

Date of
Samplin

7 12

   

Source R68. ins Index 5 C

      

      

        

  

attin 1 0.4 O 18

  

 

A comparison of standard lactose peptone broth
and tryptose lactose broth using 1 ml.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

fits of - : rec mmm colon o a
Sampling. Source Res. ing Lactose Tryptos- Index 57°C
“W ~,W;;;wmw;24 map”;'
7 12 as Pattingill 0.4 27 60 so 0 18
8
7/15/38 West Junior 0.5 40 40 20 0 251
40 60 0 424
.ilQ
7/14/38 Pattingill 1.5 32 60 so 0 _$
so 20 o 6
__ .Q
80 60 o 45
:fi
20 so 0 4
7/15/58 Moore's Park 1 200 100 100 6 116
QLEQ
100 100 0 194
__§2
100 100 0 25
1
. 100 100 o 288
7715738 west Uunior 0.7 as 60 100 0- '23
F - 21
100 40 0 209
.1. 75
0 60 0 4e
‘20 60 ‘0 ‘§§
A. 14

 

 

 

 

 

 

 

 

TABLE I (continued)

A comparison of standard lactose peptone broth
and tryptose lactose broth using 1 ml.

Cl
2 Source R03 .
,_ , ' ,_ _ _,, _:;gm--

- '*,._-_ . .1._,__.__...__.-—..._——

 
 

    
   

olon TOtal

0 I’d :: C
Tryptos: Index.3'7°C
1.1.9.... Co ‘3

     
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Moore's Park 2 100 0

165
80 100 O 58
5
7/20/38 West Junior 0.5 50 80 60 0 1210
1159
20 100 O 1792

1 5
80 100 O 1816
719

O 80 0 985
.1 has
7/20/58 Pattingill 0.3 35 0 o 0 so
' 50
2O 2O 0 4
' 95

4O 20 O 71
82
4O 4O 0 65
:l_ 85
7720/58 Moore's Park 2.5 225 40 0 55
5
80 80 4 94
_f 75
2.5 225 100 80 2 94
fig, 75
100 80 O 52
c_ 72
7721/58 Pattingill 0.4 32 0 20 0 16
' l 2
20 40 O 15
g;
0 20 0 22
20
O O 0 15
2Q
7/21/58 West Junior 0.5 20 20 40 0 13
5
O 105
133.2
0 567
2:3
20 2O 0 66
125

 

 

 

 

 

 

 

 

A comparison of standard lactose peptone broth

TABLE I (continued)

and tryptone lactose broth using 1 ml.

  

  
  
   

Date of
Sampling

Source Res.

 
 

 

 

 

Bath-
ing

Load. .4

   

fitrgptg 0 ci Iidl.':
Tryptose
_,Br9§h_-

Lactose
-EREEE_W.--

   
 
  
  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7/21/38 Moore a PerkM 2

100 60 0 8%
7/22/58 Moore's Park 1 175 100 ‘100 0 1%
i 22

100 , 100 2

-, 100 100 4

6O 20 0

7/25/58 Moore's Park 1 200 100 100 4
100 50 2 25
29
100 100 O 50
10/11/58 walterzFrench 0.6 4 0 40 0 6
60 O O ‘7‘

60 100 O

80 100 O

20 40 O

100 100 0

0 60 0

80 60 O

 

 

 

 

 

 

 

 

,TABLE II

A comparison of standard lactose peptone broth and
tryptose lactose broth using 0.5 ml. samples.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. a ‘ P ‘ Colon Total
gaggligg Source Res. ing Lactose Tryptose Index 57°C

_ p.p.m. Lo ad Bro th Bro th m

7712/68 Pattinglll 0.4 27 24 32 0 13
7726758 Pattlngill 0.4 52 0 24 0 4g
4

fir 24 32 0 83

21

7 8 0 169

1.521

16 0 16

68

7/26/58 West Junior 0.6 38 0 0 0 117

112

0 0 233

152

0 0 0 78

152

o 102

122

7/26/58 Moore's Park 1 150 52 52 2 59

84

32 8 6 120

195

7/28/38 West Junior 0.6 56 O O 419

166

0 16 0 540

476

16 16 O 1467

§§§

8 0 0 954

595

7728738 Pattingill 0.75 28 O O O 16
7

16 8 0 14

. 5

32 40 0 22

27

40 40 0 148

208

7/28/58 Moore's Park 1 175 62 4O 0 56

7

32 24 o , 104

—w __12§

40 40 0 95

 

 

£0 mmnbm

MIW
4:

g...
. N

MI was £0 mmmbm

 

 

 

 

0Q

4-:

 

GRAPH I
STREPTOGOGGI INDIGES OI LANSING
SWIMMING POOLS
USING A 5 ML. SAMPLE
"" “Tryptose lactose broth
Lactose peptone broth
/
/
/
..... ’l
o 1+ 87‘ 12 f 16 20
GRAPH I
STMP'I'OOOCGI INDICES OF LANSING
SWIMMING POOLS
USING A 0.5 ML. sum

 

__ .. __ Tryptose lactose broth

Lactose pep tone broth

 

 

S'I'P.F‘.P'T‘(‘ (".57 151T

16

21; w 32

111111an

-11+-

of the tryptose lactose broth to give a frequency curve
that is smoother than the corresponding curve of lactose
peptone broth. From the number of samples present, based
on the curve and mean Phelps index, the tryptose lactose
broth is better than the standard lactose medium with
minimal plantings. The curves suggest that this tendency
of the curve to approach the idealfrequency curve is due
to the fact that this medium may be approaching the ideal

in the detection of positive tubes.

Graph II shows the 1 ml. dilution series. The fre-
quency of the number of positive tubes is clearly shown
to be higher for the tryptose lactose broth. There also is
a lower frequency of negative samples with the tryptose
lactose medium, which would indicate that all streptococci

were detected even in minimal numbers in this dilution.

The fact that streptococci were detected even in mini-
mal numbers and in a broth with high concentration, indicates
that the streptococci found in swimmingpool waters either have
a tolerance for tryptose or that the tryptose is used easily
by them. This action would, of course, be shown in their
growth rate in the presence of tryptose, To determine if there
was an increase in growth rate several growth curves were run
comparing lactose peptone and tryptose lactose media. The
results are shown in graphs 3, H and 5. They show clearly

that the growth rate was increased, and that the lag phase

mantras

JO

TEENS

mu

GRAPH II
STREPTOCOCCI IUICES OF LANSING
SWIMMING POOLS
USING A 1 MI... SAMPLE

all

26

16

12

 

/ _ _ _.Tryptose lactose broth

 

Lactose peptone broth

 

U 20 no 60 so 100
smrococcx muons

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

000.000.0NH.H 000.00H.m¢ 000.00H ems H 0Hm ouwnm><
000+000.e0H«H 000w00m+H0 000w0mH 000 H 00m m asepaana 00\0H\m
000.000.000.H 000.000.00 000.0NH 000.H sum H
000.000.00m 000.00m.0H 000.00 00¢ H sew owesm><
000.000.00m 000q000qwmu 000.00“ 000 H [m0m m omopoeq 00\0H\m
000.000.000 000.000.sH 000.e0H 000.H new H
000.000.H00 000.msH.s0H 000.00s.m 000.0 00H.H owanm><
000+0007men 000«00H 00H 000+«mmwm 0mmqma. an H u one 0» mn\n\m
000.000.Hen 000.0HN.00H 000.¢mm.m 000.0 seH.H H. p pa
000.000.0H 000.00n.¢ 000.0n0 00H.m H00.H omsso><
000.0007m 000q00H+m 000.0e0 000 m 204W 0 smoposg 00\n\m
0 .000.0H, 000.000.e 000.MMs 000~H ,mmH.H H
000.000.00H.H0 000.000.sm 000.mm0.m 00¢.s Hs0.H run omenm><
0001000.000.0m 000.00s40e qqquwwqw 000.0 mmoqw. m «mandate 00\sm\H
000.000.000.s¢ 000.00m.0H 000.000.n 000 0 000.H H
000.000.0m0.0H 000.000.00 000.0mH.H 000.m 00N.H emsnos<
:q00H000w00H K 000.000.00. 7000 .mquan:aqqq1mxl m one o mn\sm\H
000 000 00m.0n Alm00.000.¢¢ 000. N00 . H a an
09502 NH mason o snacoa mchmoa

 

mason n. .305 o

 

 

 

emopomH wcHnmcsoo HoooOOPQonpm oHpaaoeonccoc a no mophzo Sukonw

.Hs non mauopowm no ponsdz

 

 

 

.mnpopn umOpowH omopnhup was scanned

> mamda

   

       

no spam

 

.1,

30 RnLIdVOOT

HHL

so 'smmn

VIdflLOVH

are

"It

GRATH

GROWTH CURVE OF A

cnmsialvs LACTDTF ?T°T7¥E

>

10 .00

9.00

8.00

7.00

5.00

4.00

5.00

2.0C

1.00

IE1
NON—H??03YTIC STEFPTOCOTCI /
RED

 

 

TTYPTOSF LLCTOS? U70T1

Lactose peptone broth

— - - Tryptose lactose broth

 

 

0.04

J0 HEW m I0 NHI'BIH'V‘DO'I

"IN m WIDTH:

GEAPH IV
GROWTH CURVE OF A NON-HEMOLYTIC S'EREPTOCOCCI
COMPARING LACTOSE PEPTORE AND TRYPTOSE LLC'I‘OSE BROTH

10.00.

9.00-

8.00 /

7.004

6.00

5.00, /

 

—‘ " —Tryptose lactose broth

h 00 _Lactose peptone broth

2000‘

1.00

 

0.00 0

 

fihLIdYDOT

J0

so

Vldhlaéa

hid

Y

'1

’17“ ‘2 011

Gnoqu CUPVB 0F A“"wcw_w¥w0LvTIc STRTPTOCOCCI

COWPARING LACTOSE PE7TONE AND TWYPTQSE LACT03F BROTH

10.00

9.00

8.00

7.00

8.00

5.00

4.00

 

5.00

Lactose peptone broth

 

-—- Tryptose lactose broth

1.00

 

 

0.00
0

{N
c)
to
_J
a

-15...

of the streptococci in tryptose lactose broth is shorter.

The reproduction rate was parallel for one half to one hour

at which time tryptose lactose broth increases. There was

a more or less uniform growth rate up to six hours. Then the
tryptose lactose medium began to accelerate more rapidly than
before. This is shown especially well on graph 4. At the end
of nine hours incubation the reproduction in both broths be- I
came constant and the number of organisms in the broths tend

to show parallel lines on the graphs. This increase was prob-
ably due to the fact that the first few organisms are in a
dormant condition and thus take longer to reproduce, but after
the first few generations the organism loses this dormancy and
as it was in a favorable environment starts to reproduce rapid-
ly. The tryptose lactose broth was readily used by the strepto-
cocci and therefore they grew faster in this medium while in
lactose peptone broth the organisms find themselves in an en-

vironment which they do use, but rather slowly.

As tryptose lactose broth gave favorable results with
minimal numbers of streptococci, it was decided to determine
how tryptose lactose broth would measure under routine conditions.
A number of samples were tested using the A.P. H.A. standard

methods. (2) These results are given in tables 3 and 4.

Fifty eight samples, table 3, were tested routinely and
in parallel with tryptose lactose broth and lactose peptone
broth. The tryptose lactose medium shows very clearly a higher

streptococci index than lactose peptone broth. The mean Phelps

index was 5. 73 for lactose peptone broth and 6.83 for the

TABLE III

A comparison of standard lactose peptone broth and

lactose-tryptose broth using 10 ml.

samples.

 

 

F—
_______

Date of 7

Sampling
7 20 38

 

Source

West Junior

Bath- Str Ito o 01*1 51
Lactose

ing
Lo d

33

Broth
6

Tryptose

Broth
8

Colon
Index

 

8

 

10

 

 

7720738

Pattingill

0.3

35

 

 

 

 

7720/38

Moore's Park

2.5

225

mmomemm»

 

1

0

03003030030}

 

|-'
O

10

 

10

 

7721/38

Pattingill

0.4

32

 

 

 

 

7721738

West Junior

0.5

20

10

 

OGDNOOOCD

 

10

10

12s

 

10

100

 

7721758

Moore‘s Park

150

10

10

 

 

 

 

 

10

 

10

 

OOOOOOOOOOONkOOOOOOOO

 

 

TABLE III (continued)

A comparison of standard lactose peptone broth and
lactose-tryptose broth using 10 ml. samples.

Date of
Sampling

7 22 38

_._.”

Source

 

 

Res.

Moore's Park 1

"fw

Bath 8 recto occi ndicesc lon Total
gozn Lo; d ~_

@282
460

175 48A

 

8 10 787

 

482

10 10 479

 

8 10

 

7725738

Moore's Park

'200 10 10

 

H
O

10

 

10

 

7726/58

Pattingill

32 6

 

10

 

IE3

 

h

 

7726/38

West Junior

0.6

38

5...;
H
N

 

Egan
(A
CA

 

00.6

38

E

 

OONNNNCDNG)
OODFIFOJCD

H
O
N

 

7726758

Moore‘sfiark 1.0

«t

150 1

O

10

 

as“

1

O

10

E0

 

7728738

West Junior

0.6

36

F777
H
ouo

 

10

0'
t»
O

 

10

OOOOQflOOOOOOOOONIfiOQNO
H

.p.

O) «I

x) (D

OCDOO

t

 

 

 

 

(0
OJ
.p

 

 

 

 

E

 

TABLE III (continued)

A comparison of standard lactose peptone broth and
lactose-tryptose broth using 10 ml. samples.

~‘__._.__.......— “g.

pgfé of 1 Bath =treotococc Iguic s “010 Total
Sam 11 Source Res. ing Tryptose Index 37° C
mp ng “+1 o.p.m. Load Broth Broth Count

 

 

v‘_....__—___q-——~ .—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7 28 38 Patt1ng111 0. 75 28 2 0 16
7
10 10 0 l4
5
10 10 0 22
‘ 22
10 10 0 148
“al_f 208
7728758 Moorefs Park 1.0 175 8 4 0 66
7
6 10 0 104
148
8 10 0 95
gg
10iv08 Welter French 0.6 40 2 2 0
10 ' 0 0
6 10 0
6 10 0
10 10 0
10 10 0
6 8 0
10 6 0

 

 

 

 

 

 

 

 

our:

’ ‘
t
.1. U‘

s -

{- ,1
C' 9 ) ,, 1 "“J‘fix
’ 1 1 7 Of Lin.»
‘” 1‘79 ‘ Lit"!
\ 1.
7 t E‘ ‘J w J I Jku LJL)
‘ 5T0.) " I.
v L ;— ‘ ‘
U

28

24

16

‘)
A.

 

6
4
2
o

‘BTCLS
11:1, ..
v“ Cl
STREPTOUOC

TABLE IV

A comparison of standard lactose peptone broth and

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

tryptose lactose broth using a 10 m1. sample.
33331333 sw3313fLm‘33323g‘3i3
1 16 39 Pattinglll 0.35%
10 10 O
_ s 10 o
8 0
171—7759 West Junior 1.25"J 55 2 6 o g
_* 6 6 0
'— s 6 0
1717/59 Eastern 2 2 0
2717739 walter'French 8 10 o
10 10 O
10 0
2717/59 West Junior 5 o
2717735-"2astern 2 10 0
2/51/59 Pattinglll 0.6 35 2 4 O O
8 8 0 Q
0 0 0
2 10 O
O 2 O
0 8 O
2 6 O
10 (2 samgfes) O
8 6 O
2 4 O
s 6 o __
2 2 0

 

 

 

 

 

 

 

 

TABLE IV (continued)

A comparison of standard lactose peptone broth and

Date of

tryptose lactose broth using a 10 m1.

sample.

Bath- 5 rectoWo_ci Indi. -

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-D.o m. Load , Broth Broth Count

2 27 39 Walter French 0.2 g

572/39 west Junior 1 7 6 8 O %

5/2/59 Pattingill 1 “‘ 6 s 0 175

_1 _247

372739 Eastern 1 6 8 O 8

377/39 Pattingill 1 38 10 8 O I;

10 s 0 14

14 10

10 s 0 s

1 j

s 10 0 1

.5

10 11 0 1

7

6 s 0 9

4

5/6/39 West Junior 18 4 4 0 9

5/6/59 walter fiFEnch 0.75 10 4 6 0 ‘ .1;

5 6 59 'fiatt1ng111 1 57 2“‘ 6 0 g

B76759“”E§atern 0.9 12 0 0 0 4%
3/6/59 Central 0.4 2 0 4 0

3714759 West Junior 0.5 19 s “2 0 2%

5/14/59 Walter French 0.75 52 4 0 0 3

5725/59 west Junior 0.6 31 4 0 3;

5725/59 Fattingill 0.5 50 6 0 3

23725739 walter French 0.5 15 2 0 3

5/25/59 Central 0.75 4 0 0 1%

3727/59 Eastern 0.1 15 4 s 0 ‘g

5727/59 Pattingill 0.5 54 2 4 0 8

5/27/59 walter French10.75 5s 4 4 0 8

3/27/39 West Junior 0.4 18 4 8 O 8

 

 

 

 

 

 

 

 

XOMIHCIHJ

JO

Wm
'4.)

8K

‘

"Yr"
(1

£10211

‘-
X

7‘3
155

16

 

GRAPH VII

INDICES OF LANSING

STREPTUCOCC

STIYYIWG POOLS
USING A

10 XL. CAKPLE

   

__Tryptose lactose broth

 

Lactose peptone broth

0 2 4 6 8 10

STREPTOCOCCI INDICES

-16..
tryptose lactose broth.

Using the forty six samples in table 4, the tryptose
lactose broth has an index that was higher in almost half
of the samples. The mean Phelps index was 5.35 for lactose

peptone medium and 6.39 for tryptose lactose broth.

Using the frequency of the sample index, curves for the
above series of tests were drawn. Both graphs (6 and 7) show—
ed a lower frequency of negative samples for the tryptose
lactose medium. While the lactose peptone medium had the high-
est frequency of negative samples, and it reaches its peak
frequency between an index of Oand6, but the tryptose lactose
broth peak comes between Hanle. As it has been shown that
tryptose lactose broth was more sensitive to minimal numbers
of streptococci, it was shown in the above samples that it
was equally sensitive to large numbers of streptococci, and
does not show as many negative samples as lactose peptone from

the same sample.

Stains made from lactose peptone broth showed streptococci
that were in graceful chains, while those from tryptose lactose

broth had very short chains.

Conclusions
1. Streptococci show less lag phase in tryptose lactose broth
than in lactose peptone broth.

2. Higher streptococci indices were obtained in lactose myptose

-17.,

broth from minimal plantings than in lactose peptone
broth.

Swimming pool samples tested in parallel in tryptose
lactose broth and lactose peptone broth gave higher
streptococci indices in the former broth.

The substitution of tryptose lactose broth for lactose
peptone broth for isolation of streptococci from swim-

ming pool waters is recommended.

-18..

Literature Cited

Darby, C.W. and Mallmann, W.L. Studies on Media for
Coliform Organisms. Jour. Am.W. W. Assoc. 31: 659,
1939.

Standard hethods for the Examination of Water and
Sewage, 33h. Edition; Am. Public Health Assoc.
Mallmann, W. L.‘ Streptococci as an indicator of

Pool Pollution. Am. Jour. Public Health 13: 771,
1928.

Mallmann, W. L. and Sypien, A. Pollution Indices

of Natural Bathing Places. Am. Journ.Public Health

23: 661, 193A.

Mallmann, W.L. and Ardrey, W. B. Unpublished

Material. 1939.

Klang, Marvin The significance of streptococci in
measuring the sanitary quality of a swimming pool.
Thesis for the degree of M.S. Mich State College, 1933.
Habermann, R. T. The relative importance of Strepto-
cocci, Escherichia coli, and total count as indicators
of pollution in chlorinated swimming pools. Thesis for
the degree of M.S. Mich.State College, 1935.

Prescott, S. C. and Baker, S. K. On some cultural
relations and antagonisms of Bacillus 95;; and Huston's
sewage streptococci, with a method for the detection
and separation of these microrganisms in polluted

waters. Jour._;nfect. Dis. 1: 193, 1904.

 

- 19 _

9. Todd, D. D. A New Color Medium for the isolation and

differentiation of Streptococci. iggr. Infect. 915.
7: 73, 1910.

10. Crowe, Warren H. Differentiation of the Streptococci.
Jour. Path. and Bact. 24: 361, 1921.

11. Crowe, Warren H. A differential medium for Strepto-
cocci. Jour. Path. and Bact. 26: 51, 1923.

12. Ayers, S. H. and Johnson, W.T. Jr. Studies of Strepto-
coccus. A medium for Stock cultures and other bacteria.
Jour;_§apt. 9: 111, 1924.

13. Crowe, Warren H. Differentiation of the streptococcus
by Crowe's medium. Jour. Path. and Bact. 27: 449, 1924.

14. Beckwith, T.D. and Rose, E.T. Effect of medium of rates
of multiplication, virulence and heat susceptibility

of hemolytic streptococcus. Jour. Infect. Dis. 37: 277,

 

1925.

15. Spray, R. S. A blood-clot digest medium for cultivation
of hemophilic and other bacteria. Jour. Bact. 13: 14,
1927.

16. Medalia, L. S., Bailey, K. R. and Atwood c. A Modified
Loeffler's blood serum medium useful in the routine
health department eramination for Diphtheria and
streptococcus infections. Jour. Bact. 21: 119, 1931.

17. James, W. E. A basal medium for the primary isolation
of pathogens, a quickly prepared medium including access-
ory growth factors from fish roe or quahaugs. Jour. Bact.

25: 323, 1934.

18.

19.

20.

21.

22.

23.

24.

25.

26.

- 20 -

fright, W. D. The preparation of Nutrient agar with
special reference to pneumococci, streptococci and
other gram positive organisms. Jour. Infect. Dis.
39: 359, 1934.

Chapman, G. H., Lieb, C.W., Berens, C. and Curcio, L.

The isolation of probable pathogenic staphylococci.

Jour. Bact. 33:533, 1937.

Saffor, S. C. E., Sherman, J. H. and Hodge, H. M.

Streptococcus salivarius. Jour. Bact. 33: 263, 1937.

 

Sherman, J. M. and Stark, P.‘ The fermentation of
disaccharides by Streptococcus thermophilus. Jour. Bact.
36: 77. 1938.

Rahn, 0., Hegarty, C.P., and Deuel, R. E. Factors
influencing the rate of fermentation of Streptococcus
lactis. dour, Bac . 35: 547, 1938.

Lloyd, D. J. On Vitamins, Amino Acids, and other
chemical factors involved in the growth of the mening-
ococcus. Jour. Path. andeaqE. 21: 113, 1916..

Cole, S.W. and Lloyd, D. J. The preparation of solid
and liquid media for the cultivation of the gonococcus.
Jour. Path. and Bact. 21: 267, 1917.

Kendall, I. A., Day, A.A., Walker, A.W., and Ryan, M.
The Fermentation reactions of certain streptococci.

XLll Studies in bacterial metabolism. Jour. Infect. Dis.
25: 189, 1919.

Ayers, S. H., and Nudge, 0.8. Relation of vitamins to the

jl‘ll Ill lulu-1 4‘ (I t I.. 1‘ I 1'1

 

27.

29.

30.

31.

32.

33.

35.

-21-

growth of streptococcus. Jour. Bact, 7: 449, 1922.

.Whitehead, H. R. Studies in bacterial nutrition.

IlI PhOSphates and growth of streptococci. Jour.

Biol, Chem. 20: 1147, 1926.

Hucker, G. J. Biochemical reactions produced by
streptococcus. JQEI‘,BQQI‘ 17: 6 ab., 1929.

Krashow, F., and Gies, W.J. Biochemical studies of
streptococci. Jour. Dent. Res. 9: 29, 1929

Hutner, S. H. Experiments on the nutrition of
streptococci. Jour. Bact. 35: 429, 1938.

Ayers, S. H. Hydrogen-ion concentration in cultures

of streptococci. Jour, Bact, 1: 84, 1916.

Grace, L. G. and Highberger, F. Acid production by
streptococcus viridans in mediums of different pH
concentrations. Jour. Infect. Dis. 26: 451, 1920.
Foster, L. F. Relation of hydrogen-ion concention

to growth, viability, and fermentative activity of
streptococcus. Jour. Bact. 6: 161, 1921.

Foster, L. F. and Randall, S. B. A study of the
variations in hydrogen-ion concentration of broth media.
Jour. Bact. 6: 143, 1921.

Mallmann, W. L. and Gary, W. Study of bacteriological
methods of testing and means of disinfecting water with

chlorine. Am. Jour. Public Health. 23: 35, 1939.

 

.‘.\

,1?" -

 

:3.
f4}
‘ the
r- 5‘ i
o I
(.-
2! - i
{'1'“
.p...

 

T612.9 8562 121491

Shulls

T612 . 9 8562 121491
Shulls

A study of different media.
for the detection of minimal

l

lllHlllHlll

03169 4460

ll
I'll!
II
II
I
I.
II
I
III
I]
l
III
I
III
I
ll
III
III
III

1293

JIHIHIHIHEIIIIHI