A STUDY OF VARIOUS METHODS FOR
THE ENUMERATEON OF ENTEROCOCC! IN
RHVER WATERS AND SEWAGE

Thesis for Hue Degree of pk. D.
MICHIGAN STATE UNIVERSITY

Karl Kereluk
1956

 

This is to certifg that the
thesis entitled

ASUfiyofVmfimmEbUmfisfm‘fiw
Enumeration of Enterococci in Fiver Waters and dcwage

presented bu
Karl Kereluk
has been accepted towards fulfillment

of the requirements for

Ph.D. Iicrobioloyy and

Public Health

degree in

DmCJunc 26, 1956

0-169

 

 

 

 

 

A STUDY OF VARIOUS METHODS FOR THE ENUMERATION OF

ENTEROCOCCI IN RIVER WATERS AND SEWAGE

BY
Karl Kereluk

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies
of Michigan State University of Agriculture and
Applied Science in partial fulfillment of the
requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

L~.‘\ ‘
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Year 1956 Q‘ ,
K. ‘ e-

L

Approved by gr} Ii{/XfX/\§E:$:\fvy\limggxpn I: _. .

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\

    

Karl Kereluk

A drop plate technic was introduced and a specific medium
for growing typical and atypical enterococci was developed.

The formula for this medium was as follows:

 

 

Ingredient Grams per liter
Phytone . 20.0
Lactose 5.0
Sodium chloride 5.0
Sodium azide 0.h
Yeast extract 5.0
Ethyl violet 0.00083
K2HPOu 2.7
KHZPOu 2.7
Agar 15.0
pH - 7.0

Sterilized at 121 C for 15 minutes

Samples of river and sewage waters were tested by a
most probable number procedure, drop plate, and the membrane
.filter. The drop plate method detects more enterococci than
by any method used, however, the method has its limitations
in that it is effective only in examination of high popula-
tion waters.

The membrane filter method serves amply where the limi-
tations of the drop plate method begin. A modified ethyl
violet azide medium for the enumeration of enterococci for

use with the membrane filter technic was introduced.

 

A STUDY OF VARIOUS METHODS FOR THE ENUMERATION OF

ENTEROCOCCI IN RIVER WATERS AND SEWAGE

By
Karl Kereluk

A THESIS

Submitted to the School for Advanced Graduate Studies
of Michigan State University of Agriculture and
Applied Science in partial fulfillment of the
requirements for the degree of

DOCTOR OF PHILOSOPHY
Department of Microbiology and Public Health

1956

 

 

 

ACKNOWLEDGMENTS

The author expresses his sincere appreciation to
Dr. W. L. Mallmann for his patient guidance and coun-
seling during the progress of this thesis.

Thanks is expressed to Dr. 0. Kaufmann for proof-
reading this manuscript.

The author wishes to take this opportunity to express
his gratitude to all the members of the Department of
Microbiology for their kindness and for making his work

enjoyable.

 

VITA

Karl Kereluk
candidate for the degree of
Doctor of Philosophy

Final examination, June 26, 1956, 10:00 A.M., Room 300,
Giltner Hall.

Dissertation: A Study of Various Methods for the Enumeration
of Enterococci in River Waters and Sewage

Outline of Studies:

Major Subject:. Microbiology
Minor Subjects° Mycology, Chemistry

Biographical Items:
Born: November 3, 1922, Fargo, North Dakota

Undergraduate Studies: North.Dakota Agricultural College,
lane—1950, 3.8., June 6, 1950.

Graduate Studies: Michigan State College, 1951-1952,
M.S., 1952, Professor - Dr. W. L.
Mallmann. Thesis Title: Development
of a Negative Stain for the Enumera-
tion of Ruminal Microorganisms.

Ph. D., 1956, Major Professor -
Dr. W. L. Mallmann.

Experience: Bacteriologist-in-Charge, l9h9-1950, City
Health Department, Fargo, North.Dakota;
Agent (Bacteriologist), 1950-1952, USDA,
Bureau of Dairy Industry, Michigan State
College; Graduate Research Assistant,
195h-1955, Michigan State University;
Assistant to the University Sanitarian,
1952-195h. 1955-1956, Michigan State
University.

Affiliations: Society of American Bacteriologists,
Society of Sigma Xi.

 

 

 

TABDE

1.

2.

3.

9.

10.

11.

12.

LIST OF TABLES

Drop plate counts of §, gaecalis S. zymogenes
.§. liguefaciens,'§. durans grown In mediums ’. l

andOez 0.0000000000000000

Percent light transmission at 525 mu bym-granisms
grown in various experimental base media . . . . .

Drop plate counts of organisms grown in medium
No. 1, No. 5, and Mallmann Litsky formulation . .

Drop plate counts of organisms grown in a base
medium containing various concentrations of
phy tone 0 O O O O O O O O O O O O O O O O 0

Drop plate counts of g. faecalis and §. gymogengg
.grown in a base medium.contain.ng various amounts
OflaCtoae eeoeeeeeeeeoeeeeee

 

 

Drop plate counts of §. faecalis and §. sympgenes
grown in various combinations of phytone and

laCtoae O O O O O O O O O O O O O O O O O 0

Growth of various strains of organisms in the
modified ethyl violet azide-phytone medium . . . .

M.P.N. of enterococci using azide dextrose broth
as a presumptive mediumywith Mallmann Litsky ethyl
violet azide confirmatory broth and ethyl violet
azide modified broth as confirmatory media. Com.
parison with the drop plate technic using ethyl
violet azide modified agar medium. . . . . . . . .

Percentage of confirmation of 259 colonies isolated
from.the drop plates by the Sherman standards for

enterococ01ecoeeeeoeeeeoeeoceee

Number of typical and atypical colonies present on
a single drop plate determination . . . . . . . .

Enterococci growth on EVA agar medium.and on broth
medium.using the membrane filter . . . . . . . .

Comparison of the number of enterococci grown on EVA
medium.using a white millipore membrane and a black

membrane . . . . . . . . . . . . . . . . . .

PAGE

27

28

3O

32

33

35

37

39

Al

#5

53

53

7““- —.

 

‘fi‘ 1.3:!- '

 

 

 

 

 

LIST or TABLES (Cont.)

TABLE PAGE

13. Enterococci growth on a millipore membrane and
Bac-T-Flex membrane a o a c a e e a e o a a e o 56

1h. Comparison of numbers of enterococci in river
water by membrane filter technic using Slanetz
mediumandEV‘medi-lm 000.000.00.057

 

 

 

 

 

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . .
REVIEW OF LITERATURE . . . . . . . . . . . . . . .
The Presence and Distribution of Streptococci
Classification of the Streptococci . . . . .
Streptococci as Indicators of Pollution . . .
Media Used for the Isolation of Enterococci .
Surface Plating Technic . . . . . . . . . . .

Membrane Filter Technic for the Enumeration of
Enterococci................

EXPERIPENTAL o e e e e e o e e a a o o o e a
Part I: Drop Plate M0th0d o a e a e c c o e a
Discussion . . . . . . . . . .

Part II: Membrane Filter Method . . . . . .

‘ Discu831on a e e o e o I a c e
SUWARY a a e o o e e a o e e a c e e o
BIBLIOGRAPHY o a c e o a o o o o o e a o a 0

APPENDIX I O O O O O O O O O O O O I O 0

PAGE

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13
17

19
21
21
he
RB
55
59
so
65

 

 

 

 

 

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INTRODUCTION

Coliform organisms are the only recognized test organ-
isms for the detection of sewage contamination of water and
foods. The tests for these organisms are officially made
by the United States Public Health Service and local health

agencies. The official tests are prescribed in the Standard

 

Procedure for the Analysis of Water and Sewage of the Ameri-

can Public Health Association.

 

Although the coliform group is the accepted test

organisms, they do not always give a complete picture be-
ill

\ a]

cause these organisms persist in water and soil for ex- it
tended periods of time and do not indicate recent pollution.

These organisms are present in non-contaminated soil. Fecal

and non-fecal strains of coliform bacteria cannot be differ-

entiated.

Within the past 10 years, the enterococci group has
been suggested as possible test organisms for the detection
of fecal contamination in water supplies, foods, soil and
other material. These organisms are used as indicators of
pollution on much the same basis as the coliform organisms
because they are present in feces, sewage, and known con-
taminated water but they are not present in non-polluted
and virgin soil. They do not multiply outside of the human

 

or animal body except in the presence of a rich nutrient

medium. The failure of these organisms to grow outside of
the human or animal body makes them ideally suited for the
detection of recent fecal or sewage contamination.

Mallmann and Litsky (39) demonstrated that in soil
contaminated with sewage, the most probable numbers of
enterococci were one to ten to that of the coliform organ-
isms. It was also shown that the enterococci disappeared
more rapidly from the soil while the coliform group persisted
for long periods of time.» When the typhoid bacillus was comp
pared with the enterococci, the typhoid bacillus died out‘
more rapidly. These workers believed that the enterococci
were much more indicative of recent fecal pollution than
the coliform group.

In 1950 Mallmann and Seligmann (#1) reported a new
medium, dextrose azide broth, for the detection of entero-
cocci and other streptococci. However, growth in this medium
must be confirmed for streptococci by microscopic examination.

Litsky, Mallmann, and Fifield (31) developed a confirma-
tory medium to determine the enterococci index in a manner
similar to that used for the coliform group. They proposed
a new test for the enterococci using azide dextrose broth
as a presumptive medium and an ethyl violet azide broth as
a confirmatory medium. The authors found that the dextrose

azide and ethyl violet azide media confirmed 100 to 1,000

 

times as many enterococci as did the Hajna-Perry S. F.

(18) method and the Winter-Sandholzer procedures (6h).

With the introduction of the membrane filter method by
Goetz (16) in 19h7, numerous workers quickly adapted the new
technic to the detection of the coliform group. Slanetz
gfi‘gl (55) applied the new technic to the enumeration of en-
terococci in river waters. They were able to demonstrate
with this method that the counts for the enterococci were
generally higher than those obtained by other procedures.

Mallmann, Peabody, and Broitman (to) applied the surface
plating technic of Pomales-Lebr6n and Fernandez (A6) to the
enumeration of high-population samples. Milk and polluted
waters were used in the experiment. The interesting part
of this research was that large numbers of coliform organisms
were able to grow.

The purpose of this research was to adapt the surface
plating technic for waters of gross pollution and the mem-
brane filter method in slightly polluted water for the enumer—
ation of enterococci. Present methods missed a large number

of enterococci.

 

 

=5- lmg -11

 

 

 

 

REVIEW OF LITERATURE

The Presence and Distribution of Streptococci

In 189h, Laws and Andrews (30) isolated streptococci
from.hospital sewage in England. Winslow and Hunnewell
(60) in 1902 were the first in the United States to isolate
streptococci from sewage. They also found them to be simi-
lar to those found on the hands of school children in asso-
ciation with Escherichia 221;. Houston (2h) reported in
1899 that streptococci, as well as staphylococci, were found
in large numbers in sewage. Streptococci were found in 0.1
to 0.001 ml of water from.six polluted rivers. Horrocks
(23)1901, found a high percentage of streptococci in sewage-
polluted water but no Es.22ll were detected. Broadhurst (5)
in 1915 stated that streptococci were less common in soil
than in water.

In a study on the distribution of enteric streptococci,
Ostrolenk and Hunter (h3) examined soil and feces of man,
dog, cat, mouse, guinea pig, rabbit, chicken, fly and monkey.
Fifty-one fecal samples and two soil samples were examined
using Perry's S. F. broth. The soil samples were negative
for both E, 22;; and enterococci. Forty-nine of the samples
from the 10 animals contained enterococci. Only h6 samples

contained E. coli.

 

 

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Andrews (2) examined h8 samples of hay, grass and leaves

in 1906: only two samples showed streptococci. In another
series of 18 samples, only one from.a country roadside over-
flow showed a short-chained coccus.

Winslow and Palmer (63) in 1910 isolated and differ-
entiated enterococci, using acid production in sugar, from
the intestinal tract of horses, cows, and man.

In 1912, Clemesha (10) reported the presence of strep-
tococci in polluted waters in India. Streptococci were
present in 0.001 to 0.00001 of a gram of feces. They noted
that these organisms were rare in waters which were not
heavily polluted.

Broadhurst (5) in 1915 believed that the streptococci
were neither indigenous to soil nor grains. She reported
that the streptococci were difficult to obtain from feces
of dogs, cats, and cattle; but that they were easily ob-
tained from.equine and human feces.

That enterococci however, were isolated from the normal
digestive tract of calves by Orcutt (uh) in 1926. He also
showed that the streptococci were not a homogeneous group of
organisms.

Winter and Sandholzer (6h) reported that streptococci
were not routinely found in virgin soil or in soils from
heavily wooded areas. Streptococci were always present,

however in samples of human and animal feces.

 

 

 

 

 
 

  

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Steinhouse (56) in 19h1 succeeded in isolating §.
faecalis from seven orders of the class Hexapoda.

Walter and Weaver (57), Leiniger and McCleskey (31)
and Ritter gt 5; (50), showed the presence of enterococci
in well water; they preferred the enterococci test to the

coliform test.

Classification of the Enterococci

 

Escherich (1h) in 1886 described the morphology of the
streptococci in detail and found them.to be normal inhabi-
tants of the intestinal tract of infants. The lack of a
definite classification system.caused a mass of confusion
in the literature. At present doubt exists as to what con-
stitutes an enterococcus.

Hirsh and Libman (21) in 1897 described an organism,
Streptococcus enteritis, which appeared to be identical with
the enterococcus of Thiercelin (Micrococcus avalis of Escher-

ich). He emphasized the pleomorphic morphology of the or-

 

ganism.and stated that the organism was able to grow at A6 0,
did not liquify gelatin, and inconsistently coagulated milk.
The organism was killed by an exposure for 15 to 20 minutes
at a temperature of 60 C and did not ferment sugars.

Winslow and Palmer (63) reported on the fermentative

characteristics of 116 strains of Streptococcug isolated from

 

 

 

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10 persons. The majority of the samples were from diarrheal

stools.

Gordon in 1902 (I?) started the important work of class—
ifying streptococci. He introduced seven biochemical tests
which are associated with his name. By means of these tests,
he was able to distinguish h8 varieties among 300 strepto-
cocci isolated from.nommal saliva.

The first exhaustive studies on the human fecal strepto-
cocci were made by Houston (25) in 190k. He classified 300
strains by means of the Gordon fermentations, taken from.l9
stool samples. Houston discovered that the organisms fell
into so groups on the basis of their fermentative charac-
teristics and proposed 10 classes into which the majority
of his strains fell.

Andrews and Horder (3), using the results of Gordon and
Houston, applied a series of tests to a large number of
streptococci isolated from.diseased individuals. Their re-
sults indicated that they were able to place the entire
series of organisms into seven large groups, each having a
definite type, indicated by its biological activity. The
authors were the first to describe Stggptococcug faecalis.

Donaldson (13) in 19I7summarized the characteristics
of the enterococci. He reported that the enterococci grew
in the form.of pneumococcus-like diplococci, was non-
hemolytic and produced acid from glucose, lactose, maltose,

saccharose, raffinose, glycerol, mannitol, and inositol.

 

 

Weissenbach (58) in 1918 differentiatedԤ. faecalis

from S. pyogenes using a liquid medium containing 10 percent
bile. The enterococci grew but the other streptococci did
not. I

Dible (12) in 1921 reported that the ability to with-
stand exposure to heat was not a consistent characteristic
of all intestinal streptococci. By this differentiation
he was able to divide the enterococci into two groups, one
of which consisted of organisms having fermentative reactions
corresponding toԤ. faecalis; the other group consisted of
organisms which frequently occurred in saliva.

Bagger (h) in 1926 used one percent peptone plus one
percent ox-bile for the classification of the enterococci.

Alston (1) confirmed the work of Dible. He clearly
defined a group of organisms which could be classified as
enterococci. The characteristics of this group are: (1)
heat resistant at 60 C for 10 minutes, (2) cocci, oval in
shape, occurring in pairs or short chains, (3) non-hemolytic,
and (h) able to ferment mannitol.

RHolman (22), using hemolysin production and the ability
to ferment lactose, mannitol, and salicin, as the criterian,
classified the streptococci into 16 types.

Welsh (59) in 1929 described six strains of strepto-

cocci common to human feces.

 

 

 

 

 

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Sherman, Manor and Stark (53) made an exhaustive study
on ABA cultures of S: faecalis. They found that S, faecalis,
g. durans, §_. ongenes, 51. liquefaciens would meet the fol-
lowing requirements: (I) grow at 10 C and at A5 0, (2) come
plete reduction of litmus milk, (3) grow at pH 9.6, (h) grow
in 6.5 percent sodium chloride, and (5) grow in 0.1 percent
methylene blue in skimmed milk. '

Sherman (52) in a later work indicated that the entero-
cocci could grow only at a temperature of A5 0 and in 6.5
percent sodium chloride. He used this as a basis for group

classification of the enterococci.

Streptococci as Indicators of Pollution

Houston (2A) in 1898 was the first to report on the
significance of these organisms. He concluded that strep-
tococci are of sanitary significance and indicate a.more
recent pollution than do the coliform organisms. This re-
port lead to the name "sewage streptococci of Heuston" which
was given to these organisms. I

In 190A, Houston (25) stressed the fact that strepto-
cocci as well as staphylococci, were indicative of recent
pollution by human and animal wastes.

Later Horrocks (23) supperted Houston's findings. He

found these organisms in large numbers in sewage and polluted

  
    

 

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10

waters which contained no 2. 291.1. He found that 3'3. 923;.
gradually disappeared from many specimens of sewage kept in
'the dark in an outside veranda. The organisms which persisted
were varieties of streptococci and staphylococci.

Winslow and Hunnewell (61) were the first in the United
:States to report the presence of "sewage streptococci" in sewage
in.l902. They isolated these organisms from the hands of
school children in conjunction with‘g.‘ggli and found them

to be similar to those found in Boston sewage.

 

Prescott and Baker (#5) found streptococci in 50 samples
of polluted water. Winslow and Nibecher (62) reported strep-
tococci in "unpolluted" water samples. The latter used a
direct plating method and obtained one positive sample out of
259 water samples.

Mallmann (35) in 1928 reported that streptococci are
constant indicators of intestinal pollution. In these studies
the number of organisms present in a swimming pool paralleled
the degree of pollution as indicated by the number of bathers.
He also reported that‘g.‘ggli grew slightly in water of the
pool whereas the streptococci did not.

Savage and Wood (51) in 1918 reported that when both
coliforms and enterococci were placed into a tank of water
with small amounts of organic matter, the streptococci died

out in about two weeks. The coliforms persisted for a longer

 

period of time and in some cases actually increased in number.

 

 

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11

They concluded that streptococci might be a better indicator
of pollution than the coliform.group.

Mallmann and Sypien (A2) later made a comparison of
the coliform.and streptococci indexes. Samples were taken
five feet from.the shore of a bathing beach. The results
showed that the coliform index and the total plate count
did not always respond to changes in the bathing load.

The streptococci indexes however, did show a change. They
also reported that the streptococci disappeared overnight
while the coliform organisms and total bacterial population
as determined by a plate count, showed a slight decrease.

Winter and Sandholzer (6A) confirmed the work of Mall-
mann and Sypien. They found that the coliform.organism per-
sisted in the water for a greater distance from the source
of pollution than did the streptococci.

Mallmann and Litsky (39) reported a survival of enteric
organisms in various types of soil. Using dextrose azide
broth as an enrichment medium, they could not isolate entero-
cocci from soils which were not treated with sewage. They
found that the coliform.arganisms persisted for longer
periods of time in sewage treated soils than the entero-
cocci did. The latter die out rapidly but not as rapidly
as virulent typhoid bacilli.

 

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12

In 1951, Lattanzi and Wood (29) compared enterococci
and coliform as indexes of water pollution. They found that
the coliform test for water pollution subject to limitation
although well established in the field of water bacteriology.
Using the technic of Winter and Sandholzer, they examined
samples weekly for 11 weeks during the winter months. The
results indicated that enterococci densities followed the
same pattern as that of coliform.indexes.

Litsky gt §l_(33) compared the most probable number of
coliform.bacteria and enterococci in raw sewage. They found
a positive correlation of +0.9 between the number of coliform
bacteria and enterococci in sewage from.the settling tanks
of the Amherst sewage treatment plant during the winter,
spring and summer months. The investigators also reported
that the density of coliform bacteria was approximately 13.3
times that of enterococci.

Litsky and Mallmann (32) compared the MPN of the coli-
forms and enterococci. They found a positive correlation of
+0.8A existing between the number of coliforms and entero-
cocci in sAmples taken from the Connecticut River during a
two year period. Based upon median value of all the samples
collected in this study, the density of enterococci was
approximately 7.6 times that of the coliforms.

Leininger and McCleskey (31) examined various surface

waters to determine bacterial indicators of pollution. In

 

all the waters studied, high total bacterial counts were

associated with relatively high coliform count,‘§,‘ggli
counts, and enterococci counts. They indicated that dif-
ferences between relatively clean and recently polluted water
was more strikingly shown by the enterococci test than by

the coliform test.

Walter and Weaver (57) surveyed 52 wells to determine
the value of streptococci as an index of pollution. The
numbers of coliform.and streptococci in well waters were
identical. 'For the examination of stored samples, however
the test for streptococci was slightly superior since they
never increased in number whereas the coliform count varied.

Ritter gt'gl (A9) compared the coliform.organisms with
the enterococci in 595 well waters in Kansas. The data
showed there was a positive association of the two groups
of bacteria; the chi-square test indicated non-independence
with a probability less than 0.001. The enterococci test
was preferred to the coliform test. Atypical strains of
enterococci were isolated from well water samples of good

sanitary quality as determined by the coliform.test.

Media Used for the Isolation of Enterococci

Prescott and Baker (A5) reported in 190A that when

streptococci and E, coli were grown in mixed cultures, Eb

 

coli reached a maximum growth before the streptococci. The

‘Q. ggli_however, were gradually displaced by the streptococci
in 20 to 60 hours. In several trials the streptococci com-
pletely outgrew the‘E. 391i.

Mallmann and Gelpi (38) observed a similar succession
of growth in lactose broth. After the coliforms were con-
firmed, the tubes were reincubated for A8 hours, centrifuged
and examined for streptococci by mkupscopic methods. They
also noted that if the tubes were left at room temperature
for three days after the initial incubation, a heavy sedi-
ment formed in the bottom of the test tube. This they be-
lieved was an indication of streptococci. '

Houston (26) in 1930, described a method for the iso-
lation of streptococci which is still used by the British
Ministry of Health. The original samples were inoculated
into lactose broth tubes. The tubes were incubated for 15
to 20 minutes in a 60 C water bath. The organisms were sub-
cultured on MacConky agar. The red, pin-point colonies which
appeared after A8 hours incubation at 37 C were transferred
again to lactose broth. They were then streaked on a ni-
trate agar slant. Acid production without gas from lactose
was used to indicate streptococci. A short-chain coccus in
the water of condensation on the nitrate agar slant, and the

absence of nitrate reduction confirmed the presence of

streptococci.

 

15

The first selective medium for enterococci was described
'by'Weissenbach (58) in 1918. He used sterile, filtered,
ox-bile as an inhibitory agent.

Bagger (A) added a one percent peptone to ox-bile to
promote the growth of the streptococci. Confirmation for
the streptococci was made by a heat resistance test.

In 1932 Fleming (15) found that potassium.tellurite
was inhibitory to the coliforms in a concentration of l:l5,000
but permitted enterococci to grow. Harold (19) also used
potassium.tellurite in an agar medium. The streptococci ap-
peared as small bluish-black colonies, with a peripheral
opalescence.

Sodium.azide was first used by Hartman (20) in 1937,
as an inhibitory agent against grampnegative organisms.

Since the introduction of sodium azide, many investigators
have used it in media for the detection of enterococci.

Mallmann (36) reported in 19A0 a medium using sodium
azide for the estimation of enterococci. A broth medium
made according to the formula of Darby and Mallmann (11)
containing 1 to 5,000 concentration of sodium azide was found
to support the growth of streptococci but inhibit growth of
the coliform group.

Hajna and Perry (18) used this medium.and a A5 C incu-
bation temperature for the selection of enterococci. Growth
and acid production were regarded as indicative of fecal

streptococci.

  

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16

Chapman (7) in 19AA used a medium containing tripan
‘blue, crystal violet, and sodium.tellurite. The entero-
cocci formed a dark brown or a smooth, black, slightly-
raised.colony. ‘S. salivarius produced a pale blue, opaque
colony and §_. £433.12 produced a small blue colony.

Winter and Sandholzer (6A) in 19A6 reported a procedure
for the isolation of enterococci. The method consisted of
a sodium azide-presumptive broth and a penicillin-methylene
blue sodium chloride agar medium. Confirmation of the
streptococci is made by microscopic examination and a cata-
lase test.

Later, Chapman (8) reported another medium for the iso-
lation of enterococci. This was a modification of the former
medium. It was called "mitis-salivarius agar". On this
medium.the enterococci produced a dark blue or a raised black
colony.

In 1950, Mallmann and Seligmann (A1) made a comparative
study of several media used for the detection of enterococci.
They reported that an azide dextrose broth gave a high MPN.
Positive tubes should be checked microscopically because
gram-positive rods also grew in this medium as well as the
streptococci.

Reinhold, Swern and Hussong (A8) described a plating
medium.for the isolation and enumeration of enterococci.

The medium.is based on the ability of the enterococci to

  
   

" g , , ,-.-.
7 V La" ‘ ’7’“ ."‘.- ‘ -
J " '.5 “I" "V,II ii”.'

 

 

l7

utilize sodium citrate as an available carbon source, to
convert ditetrazolium chloride to a blue diformazan and to
grow in the presence of 0.01 percent sodium azide. The
medium was used to isolate and estimate the numbers of enter-
ococci in raw milk.

Litsky, Mallmann and Fifield (31) reported a new medium
.for'the detection of enterococci in water. They designed a
selective medium, containing ethyl violet and sodium azide,
‘which is specific for the growth of enterococci from pure
cultures or from dextrose azide broth showing growth from
sewage contaminated waters. They proposed a new test in
'which dextrose azide broth is used as a presumptive test

medium.and ethyl violet azide broth as a confirmatory medium.

Surface Plating Technic

In 19A8, Reed and Reed (A7) reported a "drOp plate"
method for counting viable bacteria. The method was rela-
tively simple. A drop of a suitably diluted material delivered
by a calibrated pipette was placed on a partially dried agar
plate. Six such drops were made on an agar plate. They demp
onstrated that counts on pure cultures of bacteria made by
the drop plate method are 7 percent higher than those made
on the same culture by the pour plate method. They also stated
that the technic is less laborious and it is a little more ac-

curate with most of the species tested.

 

 

 

V
C
J. .1'
4.

’3\

 

 

 

18

Campbell and Konowalchuk (6) using the method of Reed
and Reed (#7) showed that in parallel counts made by the pour
plate method and drop plate method on raw mdlk samples, the
drop plate method gave counts which were 27 percent higher
than by the pour plate method. They suggested that this
discrepancy resulted from.the more efficient breaking up of
clumps and chains of bacteria by the dilution procedure used
in preparing the drop plate.

Females-Lebron and Fernandez (R6) in 1952 used a drop
plate method which was similar to that of Reed and Reed.
These workers used this method for the estimation of the num-
ber of various bacteria in liquids and tissues.

Mallmann, Peabody, and Broitman (to) applied the method
of PomalesoLebrbn and Fernandez to the enumeration of high—
population samples. Milk and polluted waters were used in
the experiment.

Mallmann and Broitman (37), using the drop plate method
of Pomales-Lebron and Fernandez, made a comparison of the
drop plate method and the pour plate (standard plate count)
on retail milk samples. They concluded that the 36-hour

drop plate count was comparable to the hB-hour standard plate

count.

 

Membrane Filter Technic for the Enumeration of Enterococci

The report by Goetz (16) in 19k? on the nature and use
of the membrane filters in Germany, stimulated a number of
papers in this country on the membrane filter technic for
the bacteriological analysis of water. A still greater in-
terest occurred in the membrane filter after the publications
of Clark gt‘gl (9) and Goetz (16). These investigators des-
cribed a membrane filter procedure for the detection of coli-
form and other bacteria in water. They concluded that the
.membrane filter technic had distinct advantages over the
MPN procedures of Standard Methods for the Examination 9;
Max: 8&1 Ms.” '

Slanetz, Bent, and Bartley (55) were the first to adapt
the membrane filter technic to the enumeration of entero-
cocci in water. They developed a selective medium for use
with the membrane filter. With the membrane filter technic,
the counts for enterococci were generally higher than those
obtained by other procedures.

Litsky and Shaer (3h) made a comparative study of the
MPN and membrane filter technic for the enumeration of en-
terococci. The authors used the technic of Slanetz gt a; (53).
and the MPN method of Mallmann, Litsky, and Fifield (31).
In waters of low populations, 75 percent of the samples
showed a higher count using the MPN technic than with the

membrane filter.

55 *American Public Health Association, 10th. edition,
19 .

 

 

 

 

 

 

Slanetz, Bartley and Ray (Sh) made further studies on
the membrane filter procedures for the determination of the
enterococci count in water and sewage. They reported that
by incubating the membranes on agar instead of the usual pro-
cedure of incubating the membranes on broth pads, the count
and the size of the colony increased. In 70 percent of the
samples tested, the membrane filter technic gave a higher

count than did the MPN method of Litsky g£.g; (31).

 

 

   

 

 

 

 

 

21

EXPERIMENTAL

Part I: Drop Plate Method

Sherman and Albus, Walker and Winslow, demonstrated
that the lag phase is the most critical stage in the bac-
terial growth cycle. In order to formulate a medium for a
drop plate method, a base medium first had to be selected
which would support the growth of a minimal inoculum of organ-
isms as described by Darby and Mallmann (11). The greater the
number of bacteria that survive the critical lag phase, the
better the chance that these organisms have to multiply. The
following experiments were carried out on the assumption
that the shorter the lag phase of a bacterial growth curve,
the better the medium is for a particular species.

For a base medium, the Mallmann Litsky (31) ethyl vio-
let confirmatory medium was investigated. The authors have
demonstrated that the concentration of ethyl violet dye
~(0.00083 grams per liter) inhibits the gram-positive bacilli.
The concentration of sodium azide (O.h grams per liter) was
sufficient to inhibit the growth of the gram-negative bac-
teria and still permit the growth ofԤ. faecalis. The ob-
jective of these growth curve studies was to formulate a

medium.not only for'§. faecalis, but also for §. durans,

‘§. nympgenes and‘g. liguefaciens.

 

 

 

22

The Mallmann Litsky medium, minus the peptone and carbo-
hydrate was the base medium used in a series of growth curve
studies. The first growth curve experiments were carried
out to determine a suitable peptone source. Phytone, trypti-
case, polypeptone, and phytone plus yeast extract were used.
The concentration of the peptones was two percent.“

The growth experiments were executed by using the drop
plate method of Females-Lebren and Fernandez (#6). This
seemed to be the proper step, since the medium.was being de-
signed for drop plate technic. The method lent itself to
the conservation of media and glassware.

The method used is as follows. An lB-hour culture of
.§. faecalis, which was transferred daily for three days, was

used as the test organism. The size of the inoculum ofԤ.

faecalis into the experimenta1.medium was critical, and a pre-

 

liminary count on the culture was made to determine the number
of organisms present. The Petroff-Hauser counting chamber

was used to make this determination. After the total count
was determined, serial dilutions of the culture was made in
99 ml saline dilution blanks to give from five to 25 organ-
isms per O-OH ml. The flasks containing 100 m1 of the ex-
perimental base medium.were inoculated with the appropriate
dilution of the culture. The flasks were shaken on a Boerner

shaker for five mdnutes and a sample withdrawn at the end

 

“The peptones were procured from Baltimore Biological
Laboratory, Incorporated, Baltimore, Maryland.

 

i.

It}!
lull

\3.\

 

23

of the shaking period. A 0.2 ml pipette, graduated in 0.01

ml was used to remove the sample. Duplicate aliquots of

0.01 ml, 0.02 ml, and 0.0h ml were placed on the agar plate

at six equally spaced positions. See Plate I. The counts
were made on the duplicate drops from the three different
amounts; thus the final count shown in the tables is an average
of the six counts. Counts were made at the end of 0, three,
six, nine, 12 and 15 hours. The agar plates (drop plates)

and the base medium were incubated at 37 C.

The medium.used in the agar drop plates was recommended
by Mallmann and Seligmann (Azide Dextrose Broth) but modified
by omitting the sodium azide and adding 1.5 percent agar. Be-
fore the agar plates were used, they were dried in an inverted
position with the top lid ajar for four hours in a 37 C incu-
bator. This drying of the agar allowed the drOp-sample to
penetrate into the agar medium within several minutes. The
inoculated agar drop plates were incubated at 37 C for AS
hours and counted. The drop plates were counted by viewing
under a stereosc0pic microscope. See Plate I for a typical
drop plate.

This method for growth curve studies was repeated,
'using.§. durans,‘§. zymogenes,‘§. liquefaciens. See Figure l
for a schematic drawing of the technic used.

Comparison growth experiments were conducted using the

drop plate method and a colorimeter. A larger inoculum.was

 

 

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DROP PLATE GROWTH CURVE METHOD

fl ' ~ g

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99 ML sauna lOO ML or MEDIUM {
CULTURE? BLANK

 

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26

used in the colorimeter.method. The colorhmeter used was
the Bausch and Lomb "Spectronic 20", set at 525 millimicrons.
The percent light transmission was recorded at zero hour
incubation and at the end of every three-hour period for a
total of 15 hours incubation. The results are tabulated in
Tables 1 and 2.

The media used in the experiments had the following
formulations:

Medium.No. l

 

KZHPOh 2.7 grams/liter 7
Ethyl violet dye 0.00083 7'Base
Sodium azide 0.h

Sodium chloride - 5.0 J
Phytone (BBL) 20.0

Lactose ” 5.0 pH 7.0

Medium.No. 2

Base plus

Phytone 20.0 grams/liter
Lactose 5.0

Yeast extract 5.0 pH 7.0

Medium NO 0 3

Base plus

Lactose 5 .0 grams/liter

27

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.mzmH04mmp Ha .m .mazmoozwu .m .mHueomem .m mo weapon maaqm memo

 

 

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PERCENT LIGHT TRANSMISSION AT 525 mp BY ORGANISMS GROWN IN
VARIOUS EXPERIMENTAL BASE MEDIA

TABLE 2

 

L‘k

 

Incubation Medium Medium Medium Medium
Time No.1 No.2 No.3 No.11
§_e fae 63.118

0 (hours) 100 100 100 100
3 97 96 96 98
6 95 95 91 98
9 93 90 88 98
12 88 SS 87 91
15 1+0 18 75 65
_S_. zmggenes
0 (hours) 100 100 100 100
3 96 93 97 98
6 95 91 9h 98
9 91 88 93 98
12 82 82 93 96
15 uh. no 90 87
_S_. liguefaciens
0 (hours) 100 100 100 100
3 9h 96 96 97
6 91+ 91 91 96
9 92 89 90 96
12 88 82 87 92
15 65 14.0 614. 81
_S_. durans
0 (hours) 100 100 100 100
3 98 96 96 100
6 97 91 91 98
9 9h 90 9o 97
12 82 75 88 91+
15 an. 35 85 83

 

 

   

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29
Polypeptone (BBL) 20.0 pH 7.0
Medium.No. h
Base plus
Lactose 5.0 grams/liter
Trypticase (BBL) 20.0 pH 7.0

Sterilized at 121 C for 15 minutes

An examination of the data revealed that there was
close relationship between the results of two methods in the
growth studies.

Medium No. 2 showed the shortest lag phase of the four
base media tested for all four organisms used. Medium No. 2,
containing 0.5 percent yeast extract, showed a shorter lag
phase than medium.No. 1 which did not contain any yeast ex-
tract. Medium No. 1 and No. 2 contained phytone as the pep-
tone source. The phytone demonstrated a shorter lag phase
than the other peptone sources, trypticase (Medium No. 3)
and polypeptone (Medium No. 8).

A second set of growth experiments was run to determine
the effect of lactose and to make a comparison with the Mall-
mann Litsky medium. Medium No. 1 contained 0.5 percent
lactose and Medium.No. 5 was devoid of lactose. The drop
plate nmthod was used as previously described. The data of

the second growth experiments are tabulated in Table 3. The

’
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)})’}illllll‘ll{l(l|

 

 

TABLE 3

DROP PLATE COUNTS OF ORGANISMS GROWN IN MEDIUM NO. 1,
NO. 5, AND MALLMANN LITSKY FORMULATION

:- A
AAA; fl.

 

 

Ingflation Medium No. 1 Medium No. 5 Mfiigfign
e
g. 1.360311;
0 (hours) " 10 15
3 20* 10 15
e - 1.5 25 2°
9 125 35 25
12 175 125 25
15 #00 162 62
§_. zymogenes
0 (hours) 20 20 25
3 20 ’ 30
6 - 3° "
9 32 35 50
12 1200 50 75
15 2000 75 75
s. liguefaciens
0 (hours) 30 15 ’40
3 35 25 5°
6 SS 35 #5
9 75 125 75
12 200 175 75
15 1400 125 75
g. durans
0 (hours) 15 5 20
3 20 15 20
6 60 25 20
9 125 75 SO
12 - " "
15 - " "

*0rganisms70 . 01.4. ml .

 

 

 

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d
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results indicated that the lactose aids in the reduction

of a lag phase. This held for the four organisms. The
phytone-lactose medium (Medium No. 1) and the phytone

medium minus the lactose (Medium No. 5) showed a marked re-
duction in the lag phase as compared to the Mallmann Litsky
medium. ‘

A third series of growth experiments was run to deter-
mine the amount of phytone which could be used in a medium
for the enterococci. The data are recorded in Table A. The
results indicated that an increasing phytone concentration
caused a decreasing lag phase period. A two percent concen-
tration of phytone permitted the greatest number of organisms
to grow.

To determine the amount of lactose which could be used
with the base medium containing two percent phytone, the
lactose concentration was varied. The amounts of lactose
used were: 0.5, 1.0, and 2.0 percent. The results of the
experiment are in Table 5. The highest count was observed
with §. faecalis at 0.5 percent concentration of lactose,
while §. zymogenes was the opposite. é, zympgenes gave a
higher count at the two percent lactose concentration.

A final series of growth experiments was conducted to
establish the combination concentrations of phytone and lac-
tose that would yield the shortest lag phase. The phytone

concentrations used were 0.5, 1.0, and 2.0 percent and the

 

    

  

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. . .75 .75.
A Q . T. . 2 . . LL- ;_ H- J

 

TABLE h

DROP PLATE COUNTS OF ORGANISMS GROWN IN A BASE MEDIUM
CONTAINING VARIOUS CONCENTRATIONS OF PHYTONE

Hours of Concentration of Phytone in Percent

Incubation 0 0.5 1.0 2.0

 

_S_. faecalis

 

0 10* 15 15 10 1
3 10 10 20 5 A
6 35 20 to 20 1
9 50 50 - AZ
12 50 35 100 275
15 100 125 850 950
2w 212282222

0 3o 30 25 20

3 no 20 25 50

6 50 75 50 35

9 75 50 100 175

12 150 300 u25 850

 

*0rganisms/0.0L ml.

 

 

 

DROP PLATE COUNTS OF S. FAECALIS AND S. ZYMOGENES GROWN IN A

TABLE 5

33

BASE MEDIUM CONTAINING VARIOUS AMOUNTS OF LACTOSE

 

 

Hours of Concentration of Lactose in Percent

Incubation

0.5

1.0

2.0

 

.§- faecalis

0 15* 15
3 10 10
6 110 50
9 325 250
12 1550 675
15 8200 hloo
15
Ԥ. zymogenes

o 30 25
3 30 30
6 50 125
9 100 280
12 350 925
12

*Organi ems/0. 014. m1 .

*fiMedium containing 1.5 percent dextrose and no lactose.

15

225

775
3000

950**

10
20
50
250
1125
850**

1.4

 

 

same concentrations of lactose were also used. The results

are compiled in Table 6. The results showed that the 2.0
percent concentration of phytone increased the amounts of
growth. This was demonstrated in the previous experiments.
.§- faecalis exhibited the shortest lag phase when the con-
centration of phytone was two percent in combination with a 5.
0.5 percent concentration of lactose. However,Ԥ. zymogenes 1
grew best in a 2.0 percent concentration of phytone plus a
2.0 percent concentration of lactose.

Previous experiments indicated that the yeast extract kg
shortened the lag phase for all four test organisms. The
results of these series of growth experiments indicated a
possible medium for use in a drop plate technic for the
detection of enterococci in various waters. The following

is the medium to be used in further investigations:

K HPO 2.7 grams per liter

2 1+

KHZPOu 2.7

Ethyl violet dye* 0.00083

Sodium azide 0.h

Sodium chloride - 5.0

Phytone (BBL) 20 .0

Yeast extract 5.0

Agar 15.0 pH 7.0

Sterilized at 121 C for 15 minutes

“National Aniline Division, Allied Chemical and Dye
Cor'Poration. Lot No. 12552. Dye content 57.5 percent.

 

 

 

 

TABLE 6

DROP PLATE COUNTS OF S. FAECALIS AND S. ZYMOGENES GROWN IN

VARIOUS COMBINATIONS OF PHYTONE AND LACTOSE

 

 

 

 

Hours of Phytone 0.5% Phytone 1.0% Phytone 2.0%
Incubation Lactose i Lactose Lactose
0.5% 1.0% 2.0% 0.5% 1.0% 2.0% 0.5% 1.0% 2.0%
_S_. faecalis
'0 10* 10 10 5 10 20 15 15 5
3 20 15 - 10 lo 30 10 10 15'
6 70 no - 30 30 - 110 50 -
9 62 25 50 200 75 62 325 250 225
12 200 225 75 175 200 175 1550 675 775
15 500 275 75 1250 350 560 8200 8100 3000
_8_. zmogenes
0 '30 30 25 30 20 10 30 25 10
3 - 25 50 30 20 25 30 30 20
6 35 25 50 50 125 75 50 125 50
9 75 25 125 125 175 100 100 280 250
12 325 100 175 h50 300 500 350 925 1125

*Organi sms/O .014. m1.

 

 

The medium above shall be designated as Ethyl Violet

Azide Modified or EVA modified.
In order to determine the specificity of the medium,
a number of selected test organisms were used. The EVA.modi-
fied medium was made up minus the agar and dispensed into
test tubes. The organisms were inoculated into the broth r‘
medium. The tubes were read after~h8 hours incubation at
37 C. A plus or minus sign designated growth or absence of
growth. The results are tabulated in Table 7. None of the
organisms grew except the enterococci (S, faecalis,.§. durans, g
S, liggefaciegg, andԤ. zymogenes). The phytone did not
interfere with the activity of the sodium azide or the ethyl
violet dye. The concentration of sodium azide inhibited the
growth of grampnegative organisms and the concentration of
ethyl violet dye inhibited the growth of gram-positive bacilli.
Five Red Cedar River samples and 22 sewage samples from
the East Lansing Sewage Plant were analyzed to determine the
number of enterococci. The drop plate method using EVA
modified medium, the MPN method of Mallmann Litsky and the
EVA modified broth medium.(used in place of the Mallmann
Litsky EVA confirmatory medium) were used to enumerate the
enterococci. Azide dextrose broth was used as the presump-
tive medium.in the last two cases.

The results (Table 8) showed that the EVA modified broth

medium was 100 percent more effective than the Mallmann

 

 

 

TABLE 7

 

 

GROWTH OF VARIOUS STRAINS OF ORGANISMS IN THE
MODIFIED ETHYL VIOLET AZIDE-PHYTONE MEDIUM

-_
l

Growth After

 

Species Source R8 Hours
Incubation

Aerobacter aerogenes MSU (Neu) -577
Pseudomonas aeruginosa MSU -
Sarcina citreus MSU -
Proteus vulgaris MSU -
Sarcina lutea MSU -
Chromobacter violaceum MSU‘ -
Bacillus cereus MSU (Neu) -
Bacillus megatherium MSU " -
Bacillus circulans Wayne U. -
Bacillus mesentericus Wayne U. -
Bacillus subtilis

var. aterrimus Wayne U. -
Bacillus ruber (of Kiel) Wayne U. -
Bacillus rubidum A Wayne U. -
E. coli var. mutabilis Wayne U. -
E. coli strain B Hayne U. -
E. coli MSU -
Micrococcus pyogenes var.

aureus Brail strain MSU (Neu) -
Micro coccus pyogenes var . '

aureus Wayne U. -

Micrococcus
albug pyogenes Var 0

Wayne

U.

 

 

 

TABLE 7 (Cont.)

 

Source

Growth.After

 

Species he Hours
Incubation
Micrococcus pyogenes var.
roseus Wayne U. -
Streptococcus sp. B.
hemolytic MSU (Neu) -
S. sp. B. hemolytic MSU " A -
S. sp. Lancefield E MSU " -
S. equisimilis MSU " -
s. pyogenes human A MSU '1 -
s. faecalis MSU " +**
S. durans MSU '3 +
S. liquefaciens MSU “ +
S. zymogenes MSU " +

 

*Negative, no growth

*flPositive, growth

 

 

 

 

M.P.N.

Sample Type of Sample

TABLE 8

OF ENTEROCOCCI USING AZIDE DEXTROSE BROTH AS A PRESUMPTIVE
MEDIUM WITH MALLMANN LITSKY ETHYL VIOLET AZIDE CONFIRMATORY

BROTH AND ETHYL VIOLET AZIDE MODIFIED BROTH AS

CONFIRMATORY MEDIA.

COMPARISON WITH THE
DROP PLATE TECHNIC USING ETHYL VIOLET
AZIDE MODIFIED AGAR MEDIUM

w

Mallmann Litsky EVA Modified EVA Drop Plate

 

 

No.
1 River ulo 17,000 26,300
2 Sewage 130,000 350,000 3R7,000
3 River 2,800 7,000 u,000
h Sewage 79,000 5h0,000 550,000
5 Sewage 170,000 350,000 270,000
6 Sewage 170,000 170,000 820,000
7 River 2,u00 2,h00 6,000
8 Sewage 1,800,000 2,800,000 1,110,000
9 Sewage 36,000 lh0,000 u02,000
10 River 1,100 1,800 5,000
11 Sewage 200.000 2u0,000 820,000
12 Sewage 1,800 1,800 15,000
13 Sewage 79,000 79,000 h10,000
18 River 130 700 2,000
15 Sewage 2h0,000 280.000 920,000
16 Sewage 330,000 890.000 560,000
17 Sewage 330,000 330,000 6h0,000
18 Sewage 19,000 79,000 250,000
19 Sewage 130,000 200.000 1,280,000
20 Sewage 170,000 350,000 350,000
21 Sewage 1,100,000 1,100,000 1,015,000
22 Sewage 35,000 100,000 182,000

~..__.

Log Average. 10,100 81,500 7 158,000

   
    
 

 

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Litsky EVA medium in the MPN method. The drop plate util-
izing the EVA modified medium was 98 percent higher than the
MPN of the EVA modified confirmatory medium. The results
are compiled in Table 8.

From.drop plates 259 colonies were isolated and trans-
ferred into brain heart infusion broth. The isolations were
made under a stereoscopic microscope with 10X magnification.
The tubes were incubated at 37 C for 88 hours. From the
brain heart infusion broth, transfers were made into the fol-
lowing media: brain heart infusion broth for incubation at
85 C in a water bath; 0.1 percent methylene blue in skim
milk, brain heart infusion broth with 6.5 percent sodium.
chloride, and brain heart infusion broth at a pH of 9.6,
incubated at 37 C. If the isolates grew in all four media,
the organism was considered to be a member of the enterococci
group based on the Sherman classification (52). MicroscOpic
examinations were made on the isolated organism. The results
are compiled in Table 9. 0f the 259 organisms isolated, 95.8
percent were classified as enterococci.

Two types of colonies were present on the'drop plate.
One was a round, raised white colony and the other a round,
flat, grey colony. The 95.8 percent of the colonies which
confirmed as enterococci by the Sherman criteria were white
colonies. Forty-one additional isolations were made taking

only the flat grey colonies,.and tested by the Sherman criteria

 

 

 

 

TABLE 9

PERCENTAGE OF CONFIRMATION OF 259 COLONIES ISOLATED FROM THE
DROP PLATES BY THE SHERMAN STANDARDS FOR ENTEROCOCCI

1

 

C 0.1% Broth with Broth Micros00pic

RS Methylene 6.5% Sod. at Examination -

Incubation Blue in Chloride pH Gram positive
Skim Milk 9.6 cocci in pair

or short chains

Number of

positives 258 281 282 287 259
Percentage 99.8 95.8 95.6 96.9 100

 

‘5.

U
n

. 7, éz‘finh. . ‘. 1‘
. “'95-'37“ ”in-'1.
, I I r

"I
.. “-7 a. _. ._g

  
  
 
 

      

‘\

82

for enterococci.

These colonies grew at 85 C but did not

mee t the other requirements. The microscopic examination

or these isolates revealed gram-positive cocci in pairs

or short chains. Their morphology was typically that of

the. enterococci. The 81 isolates were transferred every

12 hours for five

days in brain heart infusion broth. After 3
the

tenth transfer, the organisms were again tested by the

Stierman requirements. Four of the 81 isolates were confirmed

as enterococci. Based on these data the flat grey colonies ’ i

we re atypical or attenuated enterococci.

Discussion

Many investigators have demonstrated that the enter-

o<=c>cci can be used as indicators of fecal pollution. The

investigation indicates that the numbers of enterococci re-

covered is small as compared to the coliforms recovered.

mesa workers considered the enterococci poor indicators

or pollution. This may explain why for the past 50 years

the enterococci have not been used extensively as indicators

or Pollution. Lack of research in this may have been due to

the fact that there was no satisfactory medium. Mallmann

ELrid Litsky (31) improved the medium for determining the number

of Streptococci by adapting it to a MPN method similar to

that used for coliforms. The method used azide dextrose

 

 

 

 

 

br-o th as a presumptive medium and confirmed all the positive

azide dextrose tubes in the EVA medium for confirmation. In

the development of the Mallmann Litsky EVA confirmatory medium,

no attempt was made to study types of peptone used in the for-

mulation. Three peptone sources were chosen: phytone, poly-
pe ptone, trypticase, and a combination of phytone and yeast
eJlt‘bract. The phytone produced the highest number of organisms
in the shortest period of time.

Phytone is a papaic digestion of soya meal, containing

a. considerable quantity of carbohydrate derived from plant

133Lasue. With this in mind, the selection of the amount of

c=ar-bohydrate to be used in the medium had to be given some
thought. In these studies, dextrose and lactose were used.

Dextrose has been used in many formulations for the entero-

cOcci. Mallmann and Litsky (31) found that lactose was
egually as good as dextrose for the carbohydrate source.

A Two percent phytone was found to be a sufficient amount
or peptone in the formulation. Varying amounts of lactose
and dextrose were used with two percent phytone to find
which carbohydrate and the amount that would yield the
greatest number of organisms in the shortest period of time.
At the end of 12 hours of incubation the growth indicated
that S. faecalis multiplied best in a 0.5 percent concen-
tration of lactose. The count was 1,550 in this concentra-

tion while the) 1.5 percent concentration of dextrose gave

r
' I!
11", JIII‘J
I ' (l'ullulli'
1 ’}1’}I’1’ ll

 

 

 

a count of 275 organisms. S. zymogenes showed a different

growth response. At the end of 12 hours of growth, the
2.0 percent concentration of lactose gave a count of 1,125
°Pg anisms per 0.08 ml, while the 1.5 percent concentration
01‘ dextrose gave a count of 850 organisms per 0.08 ml. S.
411 uefaciens and S. durans failed to grow during this particu-
18.1- series of experiments and the trials were not repeated.
The enterococci in waters are of fecal origin and too
11 ttle is known of the length of survival or comparative
Iillirnbers of different groups. From the isolation of 300
colonies made from EVA modified agar drop plates, 81 were
remind to be atypical enterococci. When selecting the colonies
to be isolated for confirmation by the Sherman method, one
can usually differentiate between the typical and atypical
eEnterococci. The typical enterococci are raised white 0010-
rIiles and the atypical are flat grey colonies. The white
c<31onies were confirmed as enterococci by the Sherman method.
The atypical colonies do not confirm, but 99 percent grew at
LLS C and have the morphology of enterococci, a gram-positive
00cci in pairs or in short chains. The number of typical
ari-Ci atypical enterococci are recorded in Table 10. It can
be noted from the table that the number of typical entero-
c0001 are in the majority. However, four of the 81 original

at379:1.ca1 isolates confirmed after being transferred twice

daily for five days. The indication is that the atypical

 

 

   

 

NUMBER OF TYPICAL AND ATYPICAL COLONIES PRESENT ON A SINGLE DROP

 

TABLE 10

PLATE DETERMINATION

1+5

 

 

 

Typical Atypical Total
————\

1 7 8 11

a 59 27 86

3 9 5 It

LL 10 9 l9

5 26 18 80

6 20 10 30

7 20 8 28

8 26 15 81

9 6 8 10

lo 18 8 22

l :L 203 10 213

12 311. 15 329

1.3 9 0 9
AVer-age 56.0 10.0 65.5

 

 

enterococci are attenuated typical enterococci and need

only to be restored.
From several positive tubes of Mallmann Litsky EVA

cor11‘irmatory broth, streaks were made on EVA modified agar

me dium. After 211 hours incubation at 37 C atypical and
tirp :ical enterococci colonies were present. The ratio of

the atypical colonies to the typical colonies was one to

five. This may support the indication that the Mallmann

L1 tsky medium does not support the growth of the atypical
A number of

Organisms as well as the EVA modified medium.
8Ltypical colonies were seeded into the Mallmann Litsky EVA

The atypical organism formed a round, light purple

medifllfle
button on the bottom of the test tube. When compared to a

Pure culture of §. faecalis, which forms a compacted round,
purple button on the bottom of the tube, the atypical organ-

1 arms have a much lighter color and the round button is not
The same results were demonstrated when EVA

as compacted.
IIl-‘EDdified broth medium was used in the place of‘Mallmann

L1 tsky medium.
Ritter 2.15. a; (14.9) has isolated atypical enterococci from

good quality well water samples as shown by the coliform

test. The atypical enterococci have a definite place of im-
For tance in sanitary water supplies and further investiga-

tions should be carried out along this line.
A new medium has been described in the thesis which is

a mOdification of the Mallmann Litsky EVA medium. The EVA

 

 

i0

 

 

 

 

 

 

 

 

117

modified medium used with the drop plate method gives a

higher count of enterococci than by any of the methods used

today. The drop plate method has some distinct advantages

ove :- the time consuming MPN method used by Mallmann and

Lit sky. The method can be used in the smallest water labora-

tories. The drop plate method gives accurate counts and re-

Pr‘oducible results. In addition to higher bacteria counts

it conserves media and glassware. With the drop plate method

two water samples at three different dilutions can be made

on one petri dish. However, the method is not as success-

fill in low population waters. It is, therefore, recommended

for high population waters, such as grossly‘ polluted rivers

and sewage. Some river water samples have been successfully

eJiamined for enterococci by using a larger amount of sam;>le

than the 0.014. ml. A 0.1 ml sample can be used. Four such

8ia-l'nples can be made on one petri dish.

“hula?

 

Il‘lalllllllll‘

 

 

Part II: Membrane Filter Method

The membrane (molecular) filter, hereinafter designated
as HF, was first described by Goetz (16) in 19117. The MP
pro cedure has been extensively applied to the bacteriological
ana lysis of water. Several investigators have described the

MP procedures for the determination of coliform and other bac-.

_ ‘-__..;--

teria in water and concluded that the MF might have distinct
advantages over the MPN procedure of, Standard Methods for
the Examination of Water and Sewage. ‘ Slanetz gt 3; (55) y‘
I.°E><>rted on the use'of the MF technic to enumerate entero-
cccci in water.
The filter apparatus used in this study is shown in
Plate II and Figure 2. The apparatus consisted of a glass
f‘J-Iilnel, a fritted glass base on which the membrane rests, and
a rubber stopper by which a vacuum flask may be attached by
use of a special clamp. The membranes?! are 150 microns, or
0-005 inch thick, and composed of cellulose esters. The
membranes are white with an imprinted grid with each square
be presenting l/lOOth of the effective filtering area. The
Pox-e size of the membrane is such that it will retain particles
or 0.5 microns or greater. They were sterilized by placing
151319111 between absorbent pads, wrapped in desired numbers,
in Paper, and autoclaved at 121 C. (15 pounds pressure)

for 15 minutes. when ready for use, the membranes and pads

g

7 “Type EA, 147 mm, Millipore Filter Corporation, Watertown,
2’ M388.

 

 

 
 

u.-. -n. l .1 .
d: - , Ii-1-fl.3.... .J. n... .. f I
I! I. .4. h: ‘I’ ’ . A . .
ill 3%.. .‘W , . . _ V .
. . . “ a. .V
. a, .

 

     
     
    

ut'
mlllPoRE "neg How
nun

cents nu ' ”u

HOLDING CLAMP
PART at 3

 

 

has;

 

 

V. . - w‘Nta
V:
.43‘1

 

 

 

A. ”‘9“ “3"   «

51

   

may be easily handled by placing them.in sterile petri
dishes. The glass apparatus was covered with freezer foil
and sterilized by autoclaving at 121 C for 15 minutes.
The culture media used in the studies are the entero-
cocci medium of Slanetz (55) (see Appendix) and the EVA
agar medium to which 0.01 percent of 2,3,5 triphenyltetra- r*
zolium.chloride (TTC) was added. The TTC was made into a . P
one percent stock solution, placed in a brown bottle and
steamed for two hours. The TTC solution was added asepti-
cally to the above medium. . Li
The filtration and cultivation procedures used during
these studies were similar to those used for the determina-
tion of coliform bacteria in water by various investigators.
The various broth culture media tested were added in two ml
amounts to the absorbent pad when a broth base was used and
five ml were added when an agar base was used. The desired
quantity of water sample was filtered through the membrane
using a vacuum. The filters were then transferred directly
to the petri dishes containing the media. The petri dishes
were placed into a plastic vegetable crisper (13%" x 10%" x u%")
that closed tightly. A piece of moistened cheese cloth was
placed on the bottom of the vegetable crisper to maintain a
high relative humidity. The container of petri dishes was
incubated at 37 C. After AB hours incubation the colony

counts were made, using a stereoscopic microscope magnifying

10 times.

 

III. . .’ Iain!!!» i I. It! i
l . . , 1.1.

 

 

 

 

 

 

 

 

 

 

All previous work with the MF has been done with broth

media soaked into an absorbent pad. It was thought that the
membrane could be incubated directly on an agar medium. The
initial studies demonstrated that the membrane could be in-
cubated directly on top of an agar medium. It should be
noted here that with this procedure the membrane must be r
rolled on to the agar surface with care so as not to leave 5
air-pockets between the membrane and the agar. In Table 11

the results of the colony counts are recorded when the mem-

weer"?

branes are incubated on the agar surface as compared to that
incubated on the nutrient broth pads. The results indicate
that the membranes incubated on the former gave a slightly
higher enterococci count than did the membranes incubated
on the nutrient broth pads.
The studies on the membrane filter used a white
(type HA) membrane which were difficult to count because the
enterococci appear as white colonies. A black:HF was tried.
The black HA membrane filter was first sterilized
by autoclaving at 121 C for five minutes but it became
brittle and very difficult to handle. Some membranes were
so brittle they could not be touched without breaking. A
personal communication (28) regarding the sterilization of
the black membranes was as follows: "The filter is, of
course, composed of cellulosic-esters. By nature, the chemi—

cal bonds are not strong.....The addition of the non-

 

v- 3.9..“ d

- -»-v_'V;.

 

i
'1

 

 

 

q
'5

   

A I- '
I, I / I "
a,

| ' — 3::I' V V .‘. . ll. ‘“
. I” ‘ V I": '3' I ~ .

A} // i g ‘l l

; . I. “ .~

‘* I _‘l‘ f . 38 ‘9.
1., 2. I q! l " :- i
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'- -_ age _ __ Janna. 1 _.. t ‘ M

   

’5'}? fits? . -~ .
. 1 .. ’5» fl ‘ ..

'1 ' '
ul'

 

 

 

 

53

TABLE 11

ENTEROCOCCI GROWTH ON EVA AGAR MEDIUM AND ON

BROTH MEDIUM USING THE MEMBRANE FILTER
Sample MP on Agar Base MF on Broth Base
(No./10 ml) (No./lo m1)

River Water 1 h9h A f 391

" " 2 307 191

" " 3 163 5h

" " h 100 93

" " 5 3h 27

1' '3 6 89 70

Average 196 1A1
TABLE 12

COMPARISON OF THE NUMBER OF ENTEROCOCCI GROWN ON EVA MEDIUM
USING A WHITE MILLIPORE MEMBRANE AND A BLACK MEMBRANE

 

 

 

 

Sample No. Agar Base Broth Base
White Black White Black
Membrane Membrane Membrane Membrane
(No./S ml) (No./5 ml) (No./S ml) (No./S ml)
1 ‘ 26 ' ' 17 ‘ 3A 13
2 3h 17 8 20
3 3h 26 27 -
h 93 #3 70 #3
S 93 36 - AS
6 81 5h 70 A2

Average 60.1 32 51.8 32.6

 

  

Wino—7..- _..—._-‘—.. .. —‘E—=|_-J
9

 

 

 

SL1

flourescent dye, which for this purpose must be done in the
process of manufacture rather than afterward, seems in some
manner to alter the internal bonds of the constituent mole-
cules in the filter so that they 33g pg; be sterilized by
steam” They may, however, be sterilized either by high
voltage as in an electron accelerator Van de Graff machine,
or more commonly by ethylene oxide followed by a suitable
period of aeration in their container to obviate any inhibi-
tory action of residual ethylene oxide in the pores of the
filter."

Since there was no Van de Graff machine available at
the time, or ethylene oxide, the membranes were sterilized
by using ultraviolet light.

The white colonies on a black background were easier
to count. The results of the experiment (Table 12) indi-
cated that the black membrane had an effect on the number
of colonies that would grow. In every case the white membrane
gave higher counts than the black membrane.

Slanetz (55) reported the use of the Bac-T-Flexfi mem-
brane filter. He stated that the membrane was more flexible
and durable than other membranes used. He conducted all his
enterococci determinations using the Bac-T-Flex membranes;
however, he did not make any comparison of numbers of enter-

ococci which would grow on both membranes. This was the

 

 

*Bac-T-Flex filters (8 mm square grid markings), and S
and S absorbent pads No. u07. Supplied by Carl Schleicher
and Schuell Company, Keene, N. H.

 

 

 

 

,-—am "'fifrj.-

 

a
1

 

._. 2..

   

 

next experiment which was carried out and the results tabu-

lated in Table 13. A river water sample was used for the
comparison. EVA agar, EVA broth and Slanetz's medium were
tested on both the membranes. There was no significant
difference in the number of enterococci recovered from.the
two membranes.

A comparison of the number of enterococci in river
water was made by the membrane filter using an EVA medium
and Slanetz's medium on Bac-T-Flex membranes. The results
of 12 river samples are tabulated in Table IA. The EVA
medium.showed a greater recovery of enterococci than the

Slanetz medium.

Discussion

The introduction of MF technic offers a new method by

which organismssin slightly polluted waters could be concen-

trated on a membrane. This membrane retains the organisms

on its surface and allows nutrients to pass through its

microscopic pores, permitting growth. The method is advan-

tageous when the sample of water that is to be tested needs

to be concentrated to detect the microorganisms present.

The membranes which were incubated directly on agar

medium showed a higher colony count and larger colonies than

did the membranes impregnated with broth medium. This was

@
la J

fie...“ Th.-— ffi
_i'jr ..

1‘

 

i-m

 

TABLE 13

ENTEROCOCCI GROWTH ON A MILLIPORE MEMBRANE AND
Bac-T-Flex MEMBRANE

 

 

 

EVA Agar EVA Broth Slanetz Medium _

MF BTF MF BTF MF BTF F]

357 3&8 326 .251 268 22? gl 3

315 225 309 262 215 231 5_ J

289 321 276 211.6 262 277 EJ
Average:

327 296 303 253 2118 2&5

‘»

 

 

COMPARISON OF NUMBERS OF ENTEROCOCCI IN RIVER WATER BY MEMBRANE

TABLE 1h

FILTER TECHNIC USING SLANETZ MEDIUM AND EVA MEDIUM

 

 

Sample No. EVA Medium. Slanetz Medium
1 222 17A
2 2&0 152
3 95 52
h 92 5h
5 25 12
6 2A 2A
7 357 268
8 315 215
9 289 262
10 28k 227
11 255 23 1
12 321 277
; Average 201.6 162.1

 

 

 

 

 

. I
‘ as”? {1; x
. ._ .
' w. .

:21 _ - 1, . ~ , ‘ a .
A ‘ .. I x ‘ ' a
_a : . j 7 ‘
» z ‘ fl} ' t - A "u
I - ea " _ - _
7 . ' 2 ' I!" i E ' d- "' , ‘ ' ‘
1 1 j I" e . j . .I 1" . . l‘
. ~ \
I - e II II '! ._ , ?h v 7‘ .‘ ‘
~_ 1' q - ”‘71- an“ 1:31-5 dew a. , I! _ . '_’ l e. , .

       

58

noted by Slanetz gt gl_(5u), but no explanation for this oc-
currence has been given.

Red and pink colonies were isolated from.the membranes
grown with Slanetz medium.and the EVA medium. A total of A07
colonies were isolated from the membranes on EVA medium and
168 colonies were isolated from membranes on Slanetz medium.
All the isolated colonies were inoculated in brain heart in-
fusion broth and incubated for 2h hours at 37 C. From.the
brain heart infusion broth transfers were made into:

1) brain heart infusion broth and incubated at MS C, 2) 0.1
percent methylene blue in skim milk, 3) brain heart infusion
broth with 6.5 percent sodium chloride, A) brain heart infusion
broth at a pH of 9.6. If the organisms grew on all four media,
they were considered to be enterococci according to Sherman.

A total of 13 colonies from the EVA medium and AB colonies

from Slanetz medium.did not confirm as enterococci. The h8
colonies from Slanetz medium were white colonies which were
present on the membrane. Thus the EVA medium had a higher
percentage of organisms confirmed as enterococci than the

isolates from Slanetz medium.

 

9. T.)-..- '1 ‘
t
,~'r

if.
1Q 3;

 

E-np ‘ _ - u
i __ "H" [‘9.‘ a? ., ,Oq

   

 

'\ -: I. N.
e _'-.
'0

l
I
I

59

SUMMARY

A drop plate technic and a specific medium for growing
typical and atypical enterococci have been introduced.

After a series of transfers the atypical enterococci proved
to be typical enterococci. The atypical enterococci must,
therefore, be given consideration in the evaluation of the
sanitary quality of water. However, the drop plate method
has its limitations in that it is effective only in examina-
tion of high population waters. This method detects more
enterococci than by the Most Probable Number method described
by Mallmann and Litsky.

The membrane filter method can serve amply where the
limitations of the drop plate technic begin. An Ethyl
Violet Azide medium.was introduced which gives slightly
higher colony counts of enterococci than the method and
medium of Slanetz.

The drop plate method detects the greatest number of
enterococci in high population waters, whereas the membrane

filter method can detect enterococci in low population waters.

 

 

 

 

 

 

 

 

 

 

 

 

10.

11.

12.

13.

 

BIBLIOGRAPHY

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_Z‘HA
3:3:
‘ T

 

 

 

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i? ? ' 5y I V 7.7. l’ .— I¢ ‘
' i l‘ .' 3!" 1* . ,. u ' ‘
> ’ 7"“? , .. 6 hi. ' ‘ '7. ‘4 fig
’ 1: .‘fl ,, d-..-- . “1*“? 2." ' “*l- '- _I’I _ In I =7» .. , l -> g, I -

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51.

52.

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a???” at“ - - L‘,

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63

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_—..— _"mw‘g ~m'.

“rm.' .~

 

 

} APPENDIX

Composition of the Various

Medium.No. 1

KZHPOH

KHZPOu

 

Ethyl violet dyea
Sodium azide
Sodium chloride
Phytone (BBL)
Lactose

Medium No. 2

KZHPQH

KHéPOu

Ethyl violet dyefi
Sodium azide
Sodium chloride
Phytone (BBL)

Lactose

 

 

Yeast extract

 

*Ethyl violet dye -- National Aniline Division, Allied
No. 12552. Dye Content 57.5

Chemical and Dye Corporation. Lot
percent.

Experimental Media

2.7 grams/liter
2.7

0.00083

0.h

5.0
20.0

5.0 pH 7.0

2.7 grams/liter
2.7

0.00083

0.h

5.0
20.0.

5.0

5.0 pH 7.0

 

 

”an "k- a

 

 

Mediu

Med

 

 

 

 

 

Medium No. 3

KZHPOH

KH P0
2 h

Ethyl violet dyefi

Sodium.azide

Sodium chloride

Lactose

Polypeptone (BBL)

Medium No . 14.
KZHPOu
KH P0

2 u
Ethyl violet dyea
Sodium azide
Sodium.chloride

Lactose

Trypticase (BBL)

Medium No. 5
KZHPOH
KHZPOH
Ethyl violet dyefi
Sodium azide
Sodium chloride
Phytone (BBL)

2.7 grams/liter

2.7
0.00083
o.u
5.0
5.0

20.0 pH 7.0

2.7 grams/liter

2.7
0.00083
o.u
5.0
5.0

2000 pH 7.0

2.7 grams/liter
2.7

0.00083

0.u

5.0

20.0 pH 7.0

 

66

 

 

 

Malia

EV“

 

 

Mallmann Litsky Medium

KZHPOLL

KHZPQh

Ethyl violet dye*
Sodium.azide
Sodium chloride
Dextrose

Tryptose (Difco)

EVA Medium for the MF Technic

KZHPOH

KHZPOH

Sodium azide
Sodium.chloride
Ethyl violet dyes
Lactose

Tryptose

Slantez Medium

Tryptose
Yeast extract
Dextrose
Sucrose
KzPou

Sodium azide

67

2.7 grams/liter
2.7

0.00083

o.u~

5.0

15.0
2o.o pH 7.0

2.7 grams/liter
2.7

0.h

5.0

0.00083

15.0

20.0 pH 7.0

k percent
1

0.2

10.0

o.u
000“ PH 700-702

 

 

 

 

 

 

 

 

68

Prepare a one percent 2,3,5 triphenyl tetrazolium
chloride (TTC) solution, sterilize, and add aseptically
to the above two media Just before use to give 0.01 per-

cent final concentration.

 

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