A Study of Streptococci and Micrococci as Indicators of Pollution in Swimming Pool Water

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DOCTORAL DISSERTATION SERIES PUBLICATION: 5288 AUTHOR:

Edward Baker Seligmann Jr,, Ph. D ,, 1851 Michigan State College

TITLE:

A STUDY OF STREPTOCOCCI AND 1CROCOCC1 AS INDICATORS OF POLLUTION IN SWIMMING POOL WATER

University Microfilms, Ann Arbor, Michigan, 1953

A STUDY OF STREPTOCOCCI AND MICROCOCCI AS INDICATORS OF POLLUTION IN SWIMMING POOL WATER

By Edward Baker Seligmann Jr.

A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health

1951

A STUDY OF STREPTOCOCCI AND MICROCGCCl AS INDICATORS OF POLLUTION 15 SWIMMING POOL WATER

By Edward Baker Seligoann Jr.

AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agrioulture and Applied Solenoe in partial fulfillment of the requirements for the degree of DOCTOR 07 PHILOSOPHY Department of Baoteriology and Public Health

1961

Approved

**>v ^"V y

v

SSUv^

Edward Baker Seligmann Jr. A STUDY OF STREPTOCOCCI AMD MICROCOCCI AS INDICATORS OF POLLUTION IN SWIMMING POOL WATER ABSTRACT Growth of »treptooooo1 in aside dextrose broth wae com­ pared with growth In various modifications of this medium and Mallaann's aside broth.

The results of these comparisons

revealed that aside dextrose broth is the best medium for the isolation of streptoooooi from swimming pool water. A survey of swimming pools indicated that streptoooooi are quite prevalent, while the ooliform group is very rarely present.

However, the streptooooous index is dependant upon

the free chlorine residual in the pool.

If the free ohlorlne

is high, the streptooooous index is low. Identification of baoteria growing in tubes of aside dextrose broth lnooulated with swimming pool water indicated that buooal streptoooooi and Mlorooooous epidarmldie are the predominating baoteria.

A very low percentage of feoal

streptoooooi was found.

This evidenoe, and reports in the

literature, indicates that the pollution in a swimming pool is primarily from the nose, throat, mouth, and skin of the bathers with relatively little feoal pollution.

Therefore,

it is suggested that the oooous group, oonsistlng of Strep* tooooous salivarlus. Strep, altis. Strep, faeoalis. and M. epi* dermidis, be used as the indicators of pollution in swimming

Edward Baker Seligmann Jr* pools rather than the c o n f o r m group*

Based on log averages

of the streptooooous in&loea of samples from several pools over a long period of time a oooous index not to exoeed 15 was suggested*

The pool water Bhould oarry a free ohlorine

residual of 0*4 - 0*6 p.p.m. at all times*

REFERENCE Seligmann* Jr., E* B.

A study of streptoooooi and mioroooooi

as indicators of pollution in swimming pool water* Thesis submitted to the School of Graduate Studies of Miohlgan State College of Agriculture and Applied Soienoe in partial fulfillment of the requirements for the degree of Dootor of Philosophy in Bacteriology and Public Health* 1951*

The writer wishes to express his sincere gratitude to Dr, W, L. Mallmann, whose constant inspiration and unfailing interest and guidance made this investigation possible#

TABLE OF CONTENTS Page I* II.

Introduction

..... .........•.........

Procedure Part I

IV. V.

VI.

9

Part II

........ ................

19

Part III

........................

24

Part IV III.

1

.........

SO

Discussion

..........

S6

Conclusions

.........

39

A Proposed Bacteriological Standard for Swimming Pools

41

Literature Cited ••••••«..............

43

A STUDY OF STREPTOCOCCI AND MICROCOCCI AS INDICATORS OF POLLUTION IN SWIMMING POOL WATER When an organism is ohosen as an indicator of pollution in water it must be one which originates from the same source as the pollution, it must have a resistance equal to or greater than that of the pathogenic organisms present, and it must be incapable of multiplication in the water. In dealing with swimming pools supplied by a potable water supply the pollution originates with the bathers.

This

contamination occurs when the bathers' mouths are flushed out with pool water during swimming and when expectorations and nasal discharges enter the water.

Contamination from

the skin of the bathers also enters the water.

The occur­

rence of feoal material in a pool would be very slight. Therefore, the main source of pollution in a swimming pool would be the nose, mouth, throat, and skin surfaces of the bathers.

At the present time enteric organisms are

used as indicators of pollution.

It would seem that these

are poor organisms to indicate the type of pollution found in swimming pools. Streptococci and micrococci are found in large numbers in the normal throat as well as in fecal material and on the surface of the skin.

On the other hand, the coliform

baoteria are only found in fecal material.

This type of

pollution would not ordinarily occur if the "bathers are properly instructed in the use of a pool, A number of workers have demonstrated the disadvan­ tages of the coliform group as indicators of pollution* Savage and Wood (1) found that streptococci died slowly over a two week period in water inoculated with sewage, while Escherichia ooli multiplied in the same water and took much longer to decrease*

They concluded that the

presence of streptococci indicates recent pollution* Mallmann (£) showed that Eh coli could increase in pool water while the pool was not in use, but that strepto­ cocci decreased when the batners left the water*

He

concluded that streptococci and not coliform bacteria parallel the pollution of the pool as indicated by the number of bathers*

Mallmann and Gelpi (3) presented

further data to show that streptococci are better index organisms than are those in the coliform group*

They

demonstrated that chlorine resistant strains of E^ ooli could be isolated from chlorinated pool water.

These or­

ganisms could multiply in the pool and develop strains that may resist 0.5 p.p.m. and occasionally as high as 1*0 p.p.m* of available chlorine.

On the other hand,

Mallmann and Gelpi showed that streptococci and micro­ cocci lack chlorine resistance*

They found streptococci

and micrococci in considerable numbers when the chlorine dropped to 0.1 to 0*£ p.p.m. over a period of more than one day.

When the chlorine was raised to 0.5 p.p.m* the

cocci disappeared*

They concluded that the occurrence

of E. ooli in swimming pools does not necessarily re­ present dangerous pollution.

They further concluded

that the streptococcus and micrococcus test might be a better criterion of swimming pool safety than the present coliform test* Further research was presented by Mallmann and Cary (4)* Horwood, Gould, and Shwachman (S) felt strongly tiiat the sources of pollution in a swimming pool are the bathers* mouths and noses and that pollution of fecal origin is comparatively slight*

Therefore,

they did not believe that the coliform organisms should be used as indicators of pollution in swimming pools. They advised changing from the coliform organisms to streptococci as indicators of pollution. Mallmann (6) (7) presented further data in support of streptococci as indicators of pollution in swimming pools* The eighth edition' of Standard Methods for the Examination of Water and Sewage (8) included a method for determining the presence of streptococci in swimming pool water. France and Fuller (9) stated that in the one pool tested it was the exception rather than the rule to find coliform bacteria present, whereas streptococci were al­ ways present when bathers were in the pool. -3-

They also

determined with pure culture studies that the streptococci present were of tne "buccal rather than fecal type* VanderVelde, Mallmann* and Moore (10) by means of a simple experiment proved that tne source of pollution in a swimming pool is from the mouth and nasal cavities. They grouped bathers in the shallow end of the pool and while the bathers moved about with their heads above water* samples were taken.

The coliform and streptococcus indices

of these samples were zero*

At a designated time the

bathers commenced breathing exercises in which they had to exhale under water and during which water from the pool entered and was discharged from the nose and mouth. Sampling continued during this time.

The streptococcus

indices of these samples were relatively high while the coliform indices remained zero*

This would indicate that

the sources of pollution are the bathers1 mouths and nasal cavities* Stokes (11) reported the isolation of Staphylococcus albus from swimming pool water but did not advocate its use as an indicator of pollution. Broadhurst and Dix (IE) believed there was a need for a new standard for swimming pools*

They observed that

high plate counts could occur with swimming pool water with no accompanying increase in the pollution*

They

reported that Alkaligenes feoalis. isolated from chlorinated pools* resisted 5 p.p.m* of chlorine and could only be destroyed by 6 p.p.m* of chlorine. -4-

They also stated that

B» ooli resisted chlorination up to 1 to 1.5 p.p.m.

How­

ever, they did not suggest a better index organism. Tapley and Jennison (13) proposed the use of Neisseria catarrhalis as an indicator of pollution in swimming pools. However, the method for the isolation of this organism is tedious and not without certain sources of error. Mallmann and Seligmann (14) compared various media for determining the presence of streptococci in water end found that azide dextrose broth gave the highest index for these organisms. Ritter and Treece (15) investigated the occurrence of cocci in swimming pool water.

Using a medium developed

by them, they isolated streptococci only three times and staphylococcus eight times in twenty-six tests on pools certified by the Kansas State Board of Health.

It has

been shown by Mallmann and Seligmann (14) that the medium Ritter and Treece used, -while comparable to others avail­ able in 1948, is not as satisfactory for the recovery of streptococci as is azide dextrose broth.

This may be

why Ritter and Treece1a recovery of streptococci was so low.

They found no Streptooooous salivarius in 79 strains' -

examined which had been isolated from various pools.

Of

these strains 65.8 percent showed biological reactions typical of Strep, zymogenes and Strepi faecalis.

Twenty-six

of the 79 strains were atypical enterococci and one strain was of the viridans type.

They identified their strains

by means of type of hemolysis, growth at 10° and 45°C., in

the presence of 6,5 percent NaCl, at pH 9.6, and in milk containing 0.1 percent methylene blue. reduction of the dye in milk. run.

They also observed

Ho fermentation tests were

They concluded that streptococci are more numerous

in swimming pool water than are the coliform bacteria, but they believed the streptococcus test to be more strin­ gent than is needed. Tonney, Greer, and Danforth (16) and Tonney, Greer, and Liebig (17) compared the resistance of streptococci and otner bacteria to chlorine.

They found some resistant

strains of streptococci required 0.25 p.p.m. of chlorine for 15-30 seconds for complete kill. that

However, they found

coli required from 0.15 to 0.25 p.p.m. of chlorine,

for 15-30 seconds for complete kill while Strep, faecalis was killed in 15-30 seconds by 0.1 to 0.15 p.p.m. of chlorine. Johns (18) found that 2U ooli was killed by 42-90 p.p.m of chlorine in less than 15 seconds. Mallmann and Gelpi (3), as stated before, reported that strains of E. ooli could be developed which would resist 0.5 p.p.m. of free chlorine.

However, they found

that a Btock culture was killed very rapidly by 0.1 to 0.2 p.p.m.

They also found that streptococci could resist

0.1 to 0.2 p.p.m. of free chlorine, but were killed by 0.5 p.p.m. without developing chlorine resistant strains. Beckwith and Mosher (19) found that

coli

suspended

in tap water resisted chlorine to the extent that 0.1 p.p.m. -6-

caused no reduction after 60 minutes, while 1.0 p.p.m. caused only a 60 percent reduction after 60 minutes.

The

original inoculum ranged from 180,000 to 280,000 bacteria per ml. Butterfield, Wattie, Megregian, and Chambers (20) considered the variables of pH and temperature when testing for chlorine resistance.

Their findings agreed with earlier

work which proved that the killing power of chlorine de­ creases as the pH increases. Ritter and Treece (15) found that all streptococci were more resistant to free chlorine than Aerobacter aerogenes.

coli and

They found that Strep, salivarius

was killed in 10 minutes by 0.4-0.6 p.p.m. of free chlorine, in 5 minutes by 0.6-0.8 p.p.m., but immediately by 0.8-1.0 p.p.m.

On the other hand

ooli was killed in less than

5 minutes by 0.1-0.2 p.p.m. of free chlorine.

According

to them the enterococci are very resistant to free chlorine, withstanding even 0.8-1.0 p.p.m. for 30-60 minutes.

They

did not state the original inoculum. Work has been reviewed which indicates that the present index organisms, the coliform group, are unsatisfactory as indicators of pollution in swimming pools.

The coliform

group is found in fecal material which is not the primary source of pollution in swimming pools.

The sources are

the nose, throat, mouth, and skin of the bathers.

Strep­

tococci and micrococci are normal inhabitants of these areas and have been suggested by various workers as being

the proper indicator organisms of pollution in swimming pools*

Some workers have found that

coli can develop

a resistance to chlorine so that the presence of this or­ ganism does not necessarily indicate insanitary conditions* On the other hand the streptococci and miorococci do not develop such a resistance. demonstrated that

A number of workers have

ooli is very sensitive to chlorine

while streptococci are more resistant*

PROCEDURE

Due to the nature of the worX reported in this paper it was thought test to divide it into four parts and disouss each one separately. PART I Mallmann and Seligmann (14) have demonstrated that azide dextrose broth is the "best existing medium for the isolation of streptococci from water.

However, it is rea­

sonable to assume that another formulation might be even better for this purpose* media was made.

Therefore, a further study of

Growth in azide dextrose broth was com­

pared with growth in brain heart infusion broth in order to find out how inhibitory azide dextrose is for strep­ tococci.

Seventy-five ml. of the media were dispensed

in Erlenmeyer flasks and inoculated with a suitable di­ lution of the proper organism.

The American Type Culture

Collection Streptococcus faeoalis 6451 and Strep, salivarius 9222 were used.

Samples were removed from the flasks at

designated time intervals and plated in brain heart in­ fusion agar. An examination of the data presented in Table I shows that during the first eight hours of growth Strep, faeoalis grew slightly faster in brain heart infusion than it did in azide dextrose broth.

After 24 hours there were about

TABLE I Comparative Growth, of Streptococcus faeoalis ATCC 6451 in Brain Heart Infusion and in Azide Dextrose Broth

Humber of Bacteria per ml. Hours of Incubation

Brain Heart Infusion Broth •*

Azide Dextrose Broth

w*

0

150

140

2

225

200

4

4,400

2,000

6

200,000

48,000

8

3,500,000

500,000

24

900,000,000

80,000,000

-10-

TABLE II Comparative Growth of Streptococcus salivarius ATCC 9222 in Brain Heart Infusion and in Azide Dextrose Broth

Number of Bacteria per ml* Hours of Incubation

Brain Heart Infusion Broth

Azide Dextrose Broth

0

100

100

2

130

125

4

1,350

2,200

6

45,000

43,000

8

1,300,000

630,000

24

700,000,000

115,000,000

-11-

ten times as many cells in the former medium as there were in the latter.

However, an examination of Table II

reveals that during the first eight hours of growth Strep, salivarius grew almost as well in azide dextrose broth as it did in brain heart infusion.

After 24 hours

there were about six times as many cells in the latter as in the former. This would indicate that compared to brain neart infusion, azide dextrose broth is a very good medium for the growth of these two organisms.

However, azide dextrose

broth seemed to be a slightly more favorable medium for Strep, salivarius than for Strep, faeoalis. In an attempt to find a medium better than azide dextrose broth, growth curves of both streptococci in various modifications of Mallmann1s azide broth (21) and azide dextrose broth (Difco) were compared. ing formulae were tested: I Mallmann1s Azide Broth 2#

Tryptose lactose

0.5#

K 2HP04

0.4#

k h 2p o 4

0 .15^ > 0.5#

UaCl

0 .02#

Ha azide

12-

The follow­

II Tryptose

2%

Dextrose

0,5$

K2HP04

0.4%

KH2P04

0.15%

WaCl

0.5%

Na azide

0.02% III

Proteose Peptone #3

2%

Dextrose

0.5%

KgHPO^.

0.4%

KHgP04

0.15%

NaCl

0.5%

Na azide

0.02% IV

Azide Dextrose Broth Tryptose

1.5%

Beef extract

0.45%

Dextrose

0.75%

NaCl

0.75%

Na azide

0.02%

-13-

V Proteose Peptone #3 Beef extract

1.5$ 0.45$

Dextrose

0.75$

NaCl

0.75$

Na azide

0.02$ VI

Proteose Peptone #3

2$

Dextrose

0.75$

NaCl

0.75$

Na azide

0.02$ VII

Proteose Peptone #3

1.5$

Beef extract

0.45$

Dextrose

0.75$

Sucrose

0.5$

NaCl

0.75$

Na azide

0.02$

VIII - XI Modifications

of

medium

VII using one, two, three, and four percent proteose peptone #3.

-14

ill

Proteose Peptone #3 Yeast extract Dextrose

2$ 0.5$ 0.75$

Sucrose

0.5$

NaCl

0.75$

Na azide

0.02$ XIII

Proteose Peptone #3

2$

Yeast extract

0.5$

Dextrose

0*5%

Sucrose

0.5$

NaCl

0,5%

Na azide

0.02$ XIV

Tryptose

1.5$

Beef extract

0.45$

Dextrose

0# 75$

Sucrose

0.5$

NaCl

0.75$

Na azide

0.02$

-15-

XV Tryptose

2c /o

Yeast extract

0.5io %

Dextrose

0.5^

Sucrose

0.5^

NaCl

0

Na azide

0.02$

The final pH of all media was 6.8-7.2.

When the

growth curves in these media were compared it was found that they differed very little from one another.

However,

at no time did the amount of growth in any of the modi­ fications compare with the amount in azide dextrose troth. Streptococcus indices for three of the "best media were compared with indices obtained with azide dextrose broth.

Swimming pool water was used for the inoculum

in each case. Table III.

The indices obtained are presented in

An examination of the log averages of these

indices will show that azide dextrose broth is slightly better for the determination of streptoooooi in swimming pool water samples than are the other three media.

The

numbers of the media in the table correspond to the numbers of the media on the preoeeding pages. is azide dextrose broth.

Medium IV

Positive tests for streptoooooi

were those from tubes which contained gram positive oocci in ohains of four or more oells as determined by exami­ nations of Gram's stains of the sediment in tubes showing turbidity due to growth.

The streptococcus indices were -16-

TABLE III Streptococcus Indices of Swimming Pool Water Using Pour Different Media Bacterial Indices Samples

Media IV

IX

XIII

XV

1

79

24

33

49

2

13

0

4.5

13

3

0

0

0

0

4

0

0

0

0

5

790

490

460

700

6

700

700

490

1300

7

31

49

34

23

8

1300

1300

1300

2400

9

1300

790

1300

790

10

2400

790

490

1300

11

46

23

12

0

0

0

0

13

54

17

14

24

14

490

240

790

1300

15

790

130

230

330

Log averages

84

44

55

74

-17-

49.

33

■based on the number of positive tubes*

In many in­

stances with media IX, XIII, and XV the index as indi­ cated by visible turbidity was higher than that indicated by the presence of streptococci in the smear.

In all-

these instances the turbidity was due tp the presence of gram positive rods.

In all the swimming pool water

samples tested in azide dextrose broth gram positive rods were never found in dilutions higher than those contain­ ing streptococci.

The differences in the log averages

of the indices of these media are not significant, but it is undesirable to have gram positive rods growing in dilutions higher than those containing streptococci.

It

would seem that the nutritional properties of the mod­ ified media had been changed, as far as the gram positive rods are concerned, so that the inhibitive power of the sodium azide was decreased.

PA R T Streptococcus

II

a n d c o l i f o r m indices w e r e d e t e r m i n e d

for swimming pool water samples

c o l l e c t e d over a p e r i o d

of two ye a r s f r o m f i v e L a n s i n g high, s c h o o l pools* these pools supply*

All

operate o n t he L a n s i n g s o f t e n e d w a t e r

The c o llege p ools,

w h i c h are

operated with very

sm a l l loads and w h i c h c o n t a i n h a r d water,

f a i l e d to y i e l d

any s t r e p t o c o c c i w h e n e v e r t e s t e d and t h e r e f o r e the r e ­ sults

o b t a i n e d fr o m t h e s e p o o l s are not

survey. normal

Al l samples w e r e

i n c l u d e d in this

t a k e n w h e n the p o o l s wer e in

o p e r a t i o n w i t h the b a t h e r s

in the water.

Re c o r d s

of the par t s p e r m i l l i o n of f r e e and c o m b i n e d chlorine w e r e kept.

T h e o r t h o t o l u i d i n e a r s e n i t e test of H a l l i n a n

(22) w as used.

Also,

s e x of bathers,

nam e of the p o o l f r o m w h i c h the sample

wa s taken,

r e c o r d s w e r e k e p t o n the n u m b e r and

and the l e n g t h of tim e the b a t h e r s h a d b e e n in

the w a t e r p r i o r to sa mpling. T h e m e d i u m u s e d was t r o s e broth.

the D i f c o d e h y d r a t e d azide d ex­

The f o r m u l a t i o n of this m e d i u m is: Tryptose

1.5#

Beef extract

0.45#

Dextrose

0.7 5#

RaCl

0. 75#

R a az i de

0 02

. #

The f o r m u l a t i o n of this m e d i u m as r e p o r t e d by M a l l m a n n an d S e l i g m a n n

(14) w a s -19-

in error.

Grramfs stains of the sediment in eaoh tube showing turbidity due to growth after incubation at 37°C. for 48 hours were examined for the presence of gram positive cocci in chains of four or more*

A record of the smears

containing cocci but no chains was also kept. of 106 samples were tested in this manner*

A total

The strep­

tococcus and coliform indices were determined according to McCrady’s tables of most probable numbers (23).

The

free chlorine present in the pools at the time of sampl­ ing varied from 2.0 p.p.m. to 0.0 p.p.m.

Most of the

samples were taken during the first five minutes after the bathers entered the water. In Table IV the log averages of the streptococcus and coliform indices of samples have been classified according to the free chlorine residuals.

An examina­

tion of these data immediately shows that the incidence of coliform bacteria is very low.

Even at times when

no free chlorine was present there were very few coli­ form organisms.

However, as the free chlorine residual

decreases the streptococcus indices increase.

When

0.5 or more parts per million of free chlorine are present only occasional streptococci could be found. During the examination of the Gramfs stains from positive tubes no gram positive rods were found without the presence of cocci.

Occasionally cocci in chains of

less than four elements, in pairs, and singly were found in tubes containing no streptococci. -20-

In nearly

TABLE IV

Streptococcus and Coliform Indices of Swimming Pool Water Samples Collected at Various Free Chlorine Residuals Log Averages of Indices Streptococcus

Coliform

9

2,00

2.3

0.0

2

1.00

o . o

o • o

6

0.75

2.4

0.0

2

0.60

o o

p.p.m. of Free Chlorine

•

Number of Samples

0.0

11

0.50

5.4

0.0

3

0.40

14

0.0

14

0.35

19

0.0

19

0.20

27

0.0

12

0.15

140

0.0

21

0.10

530

1.1

4

0,05

3000

2.2

3

0.00

4100

1.1

-21-

all of these instances tubes containing streptococci were found at higher dilutions of tne sample.

When

extremely large amounts of free chlorine were present in the pool micrococci and no streptococci were found in the highest dilutions. In Table V the same samples as in Table IV are listed.

However, they are grouped according to total

chlorine rather than free chlorine.

Since the free

chlorine is the only readily germicidal chlorine and the total chlorine represents the sum of the free and com­ bined chlorine, the streptococcus indices do not corres­ pond to the total chlorine residuals.

If, for any

given total chlorine residual, the ratio of free chlorine to combined chlorine is high, the streptococcus index will be lower than if the ratio of free chlorine to combined chlorine is low.

-22-

TABLE V

Streptococcus and Coliform Indices of Swimming Pool Water Samples Collected at Various Total Chlorine Residuals Number of Samples

p.p.m. of Total Chlorine

Log Averages of Indices Coliform

11

2.00

2

0.0

6

1.50

3.5

0.0

IS

1.00

9.8

0.0

3

0.75

6.6

o . o

Streptococcus

28

0.50

28

0.0

1

0.40

23

0.0

10

0.35

6.4

0.0

6

0.30

220

0.0

21

0.20

750

1.1

4

0.15

3000

2.2

3

0.00

4100

1.1

PART III

Since not only streptococci, as determined, by their morphological appearance under the microscope, were found in the azide dextrose broth inoculated with swimm­ ing pool water, it was thought advisable to attempt an identification of the bacteria. After reviewing the literature on the classifica­ tion of streptococci and microcooci (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,) it was decided that certain carbohydrate fermentations and morphological character­ istics could be used to identify the types of strepto­ cocci and micrococci present.

Sherman (33) states that

serological methods have not proved useful tools for the classification of the viridans streptococci.

Since

micrococci were thought to be present, a method of identification had to be devised that would include these bacteria as well as streptococci.

The streptococci

in question belong to either the buccal or the fecal groups.

Fermentation of carbohydrates, litmus milk

reactions, and cell arrangement can be used to differ­ entiate the micrococci from these groups of streptococci. After comparing the fermentation reactions as described in Bergey1s Manual of Determinative Bacteriology and by other workers for all of the micrococci and streptococci listed in the sixth edition of the above manual it was

Jk

found that a combination of the fermentation reactions in glycerol, inulin, maltose, mannitol, and sorbitol along with litmus milk reactions and cell arrangement would be sufficient to identify the microoocci present as well as to differentiate between buccal and fecal streptococci. The difference between Strep, salivarius and Strep, mitis is not very pronounced.

Chapman (35) (36) has

developed a medium which will differentiate the two or­ ganisms.

He bases the differentiation on the property

of Strep, salivarius to produce large, mucoid colonies on a solid sucrose medium while Strep, mitis will pro­ duce only small pin point colonies.

This property of

Strep, salivarius was reported by Oerskov (37) and Oerskov and Poulsen (38) and further substantiated by Niven, Smiley, and Sherman (39).

The latter investi­

gators stated that the ability to produce the poly­ saccharide from sucrose appears to be correlated with the ability to ferment inulin, although the mucoid colony is not produced from inulin.

These two charac­

teristics, the mucoid colonies on sucrose agar and the ability to produce acid from inulin, are the main rea­ sons for classifying Strep, salivarius as separate from Strep, mitis.

Streptocoocus cultures when isolated and

tentatively identified were cultured on Chapman1s Mitis Salivarius agar to determine the type of colony produced. Fifteen samples taken from three pools were examined -25-

in the usual manner.

After 48 hours of incubation at

37°C. in azide dextrose broth, 0.1 ml. of the sediment from each of the positive tubes was spread over the surface of brain heart infusion agar plates.

After

incubation at 37°C. for 24 hours, isolated colonies were picked and placed in brain heart infusion broth. After incubation of these tubes, dilutions and pour plates were made to purify the cultures.

Isolated colonies were

then picked and these were carried in brain heart infu­ sion broth.

Each culture was checked in the identification

media at least twice.

Since many cultures of strep­

tococci die rapidly on culture media, the identification of the cultures had to be made as soon after the primary isolation as possible.

In a few oases cultures were lost

before they could be identified, but this loss was not significant enough to alter the frequencies of occurrence of the various bacteria isolated. The results of the identification of 132 cultures isolated from tubes of azide dextrose broth inoculated with swimming pool water are tabulated in Table VI.

Ac­

cording to these data 43 percent of the bacteria isolated were Miorocoocus epidermidis. 27 percent were Strep.mitiB. 15 percent were Strep, jsalivarius, 10 percent were Strep. faeoalis. and 5 percent were unidentified mierooocoi. Forty-two percent of the bacteria were buccal streptococci, while only 10 percent were feoal streptococci. -26-

TABLE VI Identification of Bacteria Occurring in Azide Dextrose Broth Inoculated with t a h e r of Strains

litis Salivarius

Mitis

Salivarius

Salivarius

Glycerol Inulin Maltose Mannitol Sorhitol

Pool later Litres

Milk

of Cooci

Slight Acid

Single

Acid Curd Reduction

Chains

Acid Curd Reduction

Chains

Acid Curd Reduction

Chains

leutral

Single

Unidentified

Single

Unidentified

Acid Curd Reduction

Chains

Acid Curd Reduction

Chains

faeoalis

faecalis

Records were kept of the origin of each culture in order to find out the ratio of micrococci to streptococci in all dilutions of a sample.

Table VII illustrates a

typical distribution of types when the streptococcus in­ dex was high.

The free residual chlorine was 0.05 p.p.m.

and the streptococcus index was 5,500 organisms per 100 ml. of sample.

Both buccal streptococci and micrococci

were found in the highest dilution. In a case when the chlorine was very high, 2.0 p.p.m. of free chlorine, only 1U epidermidis was found.

The

coccus index in this case was four and no streptococci were isolated from the tubes.

This seems to indicate

that !&•_ epidermidis is more resistant to free chlorine than are streptococci.

-28-

TABLE VII

Distribution of Types of Cocci in Dilutions of Swimming Pool Water in Azide Dextrose Broth. Tubes

ml. of sample per tube 10

1 1 M.e.

0.1

0.01

2 S.s.

1 S.m.

2 S.m.*

1 S.m.

3 M. e.

1 S.s.

1 S.m.

1 S.s.

1 S.m.

3 S.s.

1 M. e.

1 M.e.

2 S.m.

3 M. e.

1 S.s.

1 1 M.e.

2

3 2 S.s.

2 M. e.

2 S.m.

4 S.m.

1 M.e.

1 M.e.

1 S.s.

2 S.m.

2 M. e.

1 S.s.

2 S.s.

4

5

* S.m. - Streptococcus mitis S.s. - Streptococcus salivarius M.e. - Miorococcus epidermidis Streptococcus index - 3>300 Free Chlorine - 0,05 p.p.m. -29-

PART IV

It is logical tnat tiie occurrence of various bac­ teria in a cnlorinated pool water is related to the rela­ tive resistance of tne bacteria to chlorine as well as to the original number of cells introduced into tne wa­ ter.

A determination of the chlorine resistance of tne

bacteria isolated from tne pool water was made.

Cultures

Ef. coll isolated from sewage, Strep, faeoalis ATCC 6451, Strep, salivarius ATCC 9222, Strep, mitis isolated from the writer's throat, and

epidermidis isolated

from swimming pool water were tested in the presence of varying amounts of free chlorine.

Other cultures of the

streptococci in question isolated from swimming pool wa­ ter were compared with the above cultures and tneir re­ sistance was found to be comparable. A buffered, cnlorine-demand free water was prepared in the manner described by Butterfield, Wattie, Megregian, and Chambers (20).

Distilled water was cnlorinated and

allowed to stand for a few days.

This chlorinated water

was buffered to pH 7.0-7.1 and 200 ml. dispensed into 500 ml. Erlenmeyer flasks. and autoclaved.

These were then dechlorinated

After cooling, the pH was checked and any

flasks having a variance in pH were discarded.

The tests

were carried out at 27°C., which is the usual pool water temperature.

A known amount of chlorine was added to a

flask and the amount present in tne flask checked by means of the flash orthotoluidine test.

After five

minutes the chlorine residual in tne flask was again checked and if tnere was any divergence from the first reading, the flask was discarded.

The stock solution of

chlorine was made by bubbling chlorine gas into distilled water.

This solution was kept refrigerated in an amber

bottle.

If the residual chlorine in the water did not

change after a period of five minutes, a known number of cells was added and sampling began.

After all sampling

from the flask had ended the water was again checked for chlorine residual.

If the inoculum were of the proper

size, there was no change in the chlorine residual during the test.

If over 1000 cells per ml. were present in

the water in the flask, the chlorine residual would be reduced during the time of tue test.

The inoculum was

a 15 hour brain heart infusion broth culture of the or­ ganism in question, diluted with distilled water to 1-10,000 or 1-100,000.

One ml. of the final dilution

was then added to the flask as inoculum.

Since the ino-

cula were low in numbers of cells, it was found that plating the samplings was inaccurate.

If a larger num­

ber of cells had been used, the samplings could have been diluted to neutralize the chlorine and then plated. Therefore, one ml. of the inoculated chlorine water was added to a tube of brain heart infusion broth at each -31-

sampling time*

Samples were removed. 15, 30, and. 45

seconds and one, two, three, four, and five minutes after adding the cells.

The tubes of brotn were in­

cubated at 37°G. for 48 hours and tne presence of visi­ ble turbidity used as the criterion of growth. It was found that with inocula of about 100 cells per ml. of water in the flask the rate of kill was too rapid to record.

If over 1000 cells per ml. of water

in the flask were used tne organic matter added decreased the free chlorine during tne time of tne test.

Therefore,

inocula yielding about 500 cells per ml. of water in the flask were used. The data presented in Table VIII snow the compara­ tive resistance to free chlorine of various bacteria found in swimming pool water.

The number of cells per

ml. of tne chlorine water ranged from 500 to 650. can be seen that

It

epidermidis is the most resistant to

chlorine of the bacteria tested.

With 0.1 p.p.m. of

free chlorine complete kill was accomplished in four minutes but not in tnree.

When 0.2 p.p.m. of free

chlorine was present complete kill was accomplished in three minutes but not in two and when 0.3 p.p.m. of free chlorine was present complete kill was accomplished in one minute but not in 45 seconds.

On the other hand,

E. coli was completely killed in 30 seconds but not in 15 seconds by 0.1 p.p.m. of free chlorine. -32-

It was killed

TABLE VIII

Chlorine Resistance of Five Bacteria Pound in Swimming Pool Water Survival

M. epidermidis

Strep, faeoalis

Strep, salivarius

3

3-

2

-

2

2-

1

-

A

_

1 A 4

Ait J

* *

Time

3 -

in Minutes

4

A

2

0.3

0.2

0.1

0.3

0.2

0.1

p.p.m. of free chlorine

0.3

0.2

0.1

c coU

cv o c

I

i r

to

PlIi;

of free

H

chlorine

I

p

co

g p. Strep. mitis

rH • O CVJ •

C

to c

1 I CaJ Survival Time in I'inutes

Pj’vf HjOJ

r li'

completely in less than 15 seconds fcy 0,2 and 0,3 p*p*m. of free chlorine*

-35'

DISCUSSION An attempt was made to improve upon azide dextrose ■broth for tne recovery of streptococci from swimming pool waters.

It would appear that when certain modifications

were made in the medium the sodium azide would lose some of its inhibitory power toward gram positive rods.

This

would allow these rods to grow in higher dilutions than would the streptococci.

None of the modified media would

grow more of the viable streptococci than azide dextrose broth.

Therefore, with the present knowledge of media

it seems that tne medium to be used for a determination of tne streptococcus index of a swimming pool is azide dextrose broth. The failure to find any gram positive rods without tne presence of cocci in many hundreds of Gram’s stains made from positive tubes of azide dextrose broth ino­ culated with swimming pool water, indicates tnat this medium may be used without a microscopic examination. Therefore, in the determination of tne coccus index of a swimming pool a positive tube of azide dextrose broth would be one containing visible turbidity due to growth. Micrococci and. streptococci are the only bacteria found growing in all dilutions of a sample of swimming pool water inoculated into azide dextrose broth. coccus present was found to be -36-

The micro­

epidermidis.

This

organism was shown to be very resistant to chlorine both in laboratory tests and in the pool.

Since M.

epidermidis is found on the skin and mucous membranes, its presence would indicate pollution from these areas. Identification of the bacteria found in azide dextrose broth inoculated with swimming pool water shows that they are primarily of the buccal cavity and skin, while only very few represent fecal material.

This means

that the pollution entering the pool is primarily buc­ cal rather than fecal in origin.

Index organisms

should be chosen which originate from the buccal cavity. The indicator organisms in use at the present time, the coliform group, are fecal in origin.

It is obvious

that they will not indicate buccal contamination.

On

the other hand, streptococci and micrococci are found in all of the areas from which pollution in a pool may arise.

They are found in large numbers in the mouth,

throat, nose, and on the skin of bathers, as well as being found in fecal material.

Furthermore, it has

been shown that the streptococci and miorooocoi in question are more resistant to free chlorine than is E. ooli. It is proposed that the group of index organisms for swimming pool pollution be called, the "coccus group". This group would include Strep, salivarius, Strep, mitis. Strep, faeoalis, and

epidermidis. i

-37-

If the free cnlorine residual as recommended at present is maintained in tne pool, that is 0.4 to 0.6 p.p.m. of free chlorine as measured by the orthotoluidine arsenite test, then a coccus index of less tnan lb per 100 ml. of water sample should be acceptable. standard should be easy to maintain.

Such a

If the free chlorine

drops below 0.4 p.p.m. at any time it will be impossible to maintain this standard.

It is necessary to emphasize

the importance of free chlorine.

Combined chlorine is

not readily germicidal and if total chlorine is measured a certain percentage of this residual will not be readily germicidal.

It has been shown tnat tne level of the coc­

cus index is dependent upon the free chlorine in the wa­ ter and not upon tne total chlorine. A standard of a coccus index of less tnan 15 is a standard for convenience.

It is in itself rather nigh,

and properly managed pools should operate with a much lower incidence of cocci.

Examples of this are the

college pools which very seldom contain any cocci.

How­

ever, it has been found that not all pools are operated in this manner.

Therefore, an index of 15 will be exact­

ing, but it is not the lowest figure possible to maintain.

-28-

COCCLUSIONS The data indicate that azide dextrose broth should be used in the determination of the cocous index of swimming pool water. It was found that positive tubes of azide dextrose broth inoculated with swimming pool water are those showing visible turbidity due to growth. It was found that in a pool supplied with potable water the pollution is buccal in origin y/ith the pos­ sibility of occasional fecal pollution. Both micrococci and streptococci were found to be present in tne water.

Since the types found origi­

nate from the bathers, they both are indicators of pollution. The data indicate that the coccus group should be used as indicators of pollution in swimming pool water.

This group includes Strep, salivarius t Strep,

mitis, Strep, faecalis, and It has been shown that

epidermidis. coli is less resistant

to free chlorine than any of the members of tne coccus group.

This organism was only present when the free

chlorine residual was very low and at such times the coccus indices were over 500. The data indicate that free chlorine is the only readily germicidal chlorine.

It seems that 0.4 to

0.6 p.p.m. of free chlorine should, be maintained at all times in a pool.

40—

A PROPOSED BACTERIOLOGICAL STANDARD FOR SWIMMING POOLS Sampling:

Samples of the water for bacteriologi­

cal testing should, be taken while bathers are in tne water during the peak load.

Sodium thiosulfate bottles

should be used for sampling. Bacteriological examination of the sample:

Azide

dextrose broth should be inoculated with suitable di­ lutions of the swimming pool water sample.

Positive "

tubes will be those showing visible turbidity due to growth at 35-37°C. after incubation for 48 hours.

The

actual coccus index should be computed from McCrady’s table of most probable numbers.

The coccus index

should not exceed 15. Double strength azide dextrose broth should be used when 10 ml. portions are planted per tube.

When

10 ml. of tne sample are added to a test tube contain­ ing 10 ml. of a double strength broth the resulting medium will be of the proper strength.

A suggested

procedure for determining the coccus index is to inoc­ ulate five tubes of double strength azide dextrose broth with 10 ml. portions of the sample and also inoculate five tubes of single strength azide dextrose broth with 1 ml. portions of the sample. -41-

Incubate ail

tubes at 35-37°C. for 48 hours and. then observe them for visible turbidity due to growth.

If four out of five

of the 10 ml. portions are positive the coccus index will be 13.

If more tubes are positive additional samples

should be tested in order to determine whether the con­ dition is persistent.

If samples continually yield in­

dices in excess of 15 then the operation of the pool should be questioned. In order to maintain the pool in the proper sani­ tary condition from 0.4 to 0.6 p.p.m. of free chlorine should be present at all times.

42-

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The vitality and

viability of streptococci in water.

Jour.

Hyg., 16:227, 1917. Mallmann, W. L.

Streptococcus as an indicator

of swimming pool pollution.

Am. Jour, of

Public Health, 18:771, 1928. Mallmann, Y/. L. and A. G. Gelpi Jr.

Chlorine

resistance of colon bacilli and streptococci in a swimming pool.

Mich. Eng. Ex. Sta.

Bull. Ho. 27, 1930. Mallmann, W. L. and W. Cary Jr.

Study of bac­

teriological methods of testing and means of disinfecting water with chlorine.

Am.

Jour. Public Health, 23:35, 1933. Horwood, M. P., B. S. Gould, and H. Shwachman. Indices of the sanitary quality of swimming pool waters.

Jour. Am. Water Works Assoc.,

25, Ho. 1, Jan., 1933. Mallmann, W. L. pool waters.

Bacterial quality of swimming Ann. Report Div. Vet. Sci.,

Mich. State College, p. 59, 1935. Mallmann, W. L.

Standard methods for swimming

pool operation.

Presented at 37th Ann. Mich.

Conference on Bathing Places, May 1939.

Standard Methods for the Examination of Water and Sewage.

Am. Public Health Assoc.,

Eighth Edition, 1936. France, R. 1. and J. E. Fuller.

Coliform bac­

teria and streptococci in swimming pool water.

Am. Jour. Public Health, 30:1059,

1940. Vandervelde, T. L., W. L. Mallmann, and A. V. Moore.

A comparative study of chlorine and

bromine for swimming pool disinfection.

The

Sanitarian, Sept.-Oct., 1948. Stokes, W. R.

A search for pathogenic bacteria

in swimming pools.

Am. Jour. Public Health,

17:334, 1927. Broadhurst, J. and A. Dix.

Should bacterial

standards for swimming pools be revised? Jour. Bact, 15:23, 1928. Tapley, 0. 0. and M. W. Jennison. pool sanitation:

Swimming

Neisseria oatarrhalis

as an index of pollution.

A Symposium on

Hydrobiology. Univ. of Wisconsin Press, p. 355, 1941. Mallmann, W. L. and E. B. Seligmann Jr.

A

comparative study of media for the detection of streptococci in water and sewage. Jour. Public Health, 40:286, 1950.

Am.

Ritter, C. and E. L. Treece. cance of cocci in swimming

Sanitary signifi­ pools.

Am,

Jour. Public Health, 38:1532, 1948. Tonney, F. 0., F. E. Greer, and T. F. Danforth. The minimal "chlorine death points" of bac­ teria.

Am. Jour. Public Health, 18:1259,

1928. Tonney, F. 0., F. E. Greer, and G. F. Liebig. The minimal "chlorine death points" of bac­ teria.

II. Vegetative forms.

bearing organisms.

III. Spore-

Am. Jour. Public Health,

20:503, 1930. Johns, C. K.

The speed of germicidal action of

chlorine compounds upon bacteria commonly occuring in milk.

Scientific Agriculture,

10:553, 1930. Beckwith, T. D. and J. R. Mosher.

Germicidal

effectiveness of chlorine, bromine, and iodine.

Jour. Am. Water Works Assoc., 25,

No. 3, March 1933. Butterfield, C. T., E. Wattie, S. Mergregian, and C. W. Chambers.

Influence of pH and

temperature on the survival of coliforms and enteric pathogens when exposed to free chlorine. 1943.

Public Health Reports, 58:1837,

Mallmann, W. L.

A new yardstick for measuring

sewage pollution.

Sewage Works Jour,, 12,

No, 5, Sept, 1940, Hallinan, F, J,

Tests for active residual

chlorine and chloramine in water.

Jour.

Am, Water Works Assoc., 26:296, 1944, McCrady* M. H.

Tatles for rapid interpretation

of fermentation-tuhe results.

The Public

Health Jour. (Canada), 9:201, 1918. Broadhurst, J,

Enviornmental factor studies of

streptococci. Oppenhiem, C. J.

Jour. Inf, Dis., 17:277, 1915. Human fecal streptococci. Jour,

Inf. Bis., 26:117, 1920. Hucker, G. J,

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classification of genus Mioroooccus Cohn. N. Agr. Exp. Sta. Tech. Bull. No. 102:5, 1924. Holman, W. L.

The classification of streptococci

Jour. Med. Res., 54:577, 1926. Breed, A. F.

Micrococci present in the normal

cow's udder.

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Bull. 155:5, 1928. Welch, H.

Classification of the streptococci

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Jour. Bact., 17:415, 1929.

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Jour. Bact., 52:41,

31.

Safford, C. E., J. M. Sherman, and H. M. Hodge. Streptococcus aalivarius.

Jour. Bact.,

33:263, 1937. 32.

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1937. 33.

Sherman, J. M.

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35.

Chapman, G-. H.

The isolation of streptococci

from mixed cultures.

Jour. Bact., 48:113,

1944. 36.

Chapman, G. H.

The isolation and testing of

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^Oerskov, J.

Untersuchungen uber einen exzessiv

Polysaccharid-bildenden Streptokokkus. Zentr. Bact., Orig., 119:88, 1930. 38.

Oerskov, J. and.Poulsen, K. A.

Das haufige

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Zentr. Bakt., Orig., 120:125, 1931. -47-

39.

Niven, G. F., Jr., K. L. Smiley, and J. M. Sher­ man.

The production of large amounts of a

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-48