STUDY OF ENTHROCOCCI IN MILK

By
Isnwar Gopal Chavan

A TH jiSIS

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

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AM ABSTRACT

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30 i l w i M and high t a y a ^ tmw, ahart-tlao paatourlaatlaa at 77.* C
far 16 aananrta anaglaWly daatragr aataraeaaal la allk.
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atadlaa with para n l t a a s of ftr. faaaallla.

3tr. llm f N i— » . |t£. aygaaaaag and 8tr. darana rawaal that tha Majority
of tha ontaroaaaci ara auaaaptihla to praaaat paataarlaatlaa laaparataraa
tout a — 11 raalataat alaorlV ranrlTi long aaqpoaara parloda.
Tha pradaodLaaat atohgroup of aatarooaaal oeourrlag la ailk i - a t .

— n«.

DEDICATED TO
D r . Walter LeRoy Mallmann - An
inspiring teacher and sincere friend
with an ideal philosopi^r for
directing graduate studies.

AC KImOWLEDGL-IENTS

The author wishes to express hi3 deep and sincere feeling of
gratitude to Dr. W. L. Mallmann, Professor of Bacteriology and Public
health, under whose lofty inspiration, constant supervision and un­
failing interest this investigation was undertaken. He is also in­
dented to him for his valuable help in preparing the manuscript.
Grateful acknowledgment is made to Mr. M. L. Gray for taking the
photomicrographs. Thanks are expressed to D rs . H. J. Stafseth, Head
of the Department of Bacteriology, G. M. Trout, Research Professor of
Dairying, E. P. Reineke, Professor of Physiology and Pharmacology,
C. K. Smith, Department of Bacteriology and Mr. C. W. Fifield, Depart­
ment of Bacteriology for their guidance, helpful suggestions and
assistance .
The author wishes to take this opportunity to express his grati­
tude to all the members of the Department of Bacteriology for their
kindness ano for making liis work enjoyable.
The writer deeply appreciates the financial support from the
Government of Bomoay, India, and the scholarship provided oy Micliigan
State College wliich enabled him to complete this investigation.

ISHWAR GOPAL CHAVA im
Candidate for the degree of
DOCTOR OF PHILOSOPHY

Final Examination*
Dissertation*

February 2 k , 1953, 9*00 A. 14., Room 101, Giltner Hall

A study of enterococci in milk.

Outline of Studies s
Major subject*
Minor subjects*

Dairy Bacteriology
Dairy Manufacture, Physiology

biographical Items:
Born, March 6, 1 9 2 k Harli-Khurd, Bombay Province, India.
Uncergraduate studies
Rajaram College, Kolhapur, India 19lxli-19lx5.
College of Agriculture Poona,
University of Bombay, India 191x5-191x5, b. Sc. ( g r. ) . Hons.
Graduate studies, Micliigan State College, East Lansing
191x9-1953.
Experience*

County Agricultural officer, Department of Agriculture

Gadhinglaj, India 19lxb-19lx9, Agricultural officer, Model farm,
Kolhapur, India, 191x9, Predoctorate Alumni Research Fellowship,
Michigan State College, East Lansing, 1952-1953.
Member of The Society of American bacteriologists.

TABLE Ob* CONTENTS
PAGE
INTRODUCTION.........................................................

1

HISTORICAL BACKGROUND...............................................

U

EXFEnlMENTAL PROCEDURE.................................;............

21

Raw m i l k........................................................

21

Direct microscopic examination of raw milk for total bacterial
count..............

21

Standard plate count for raw and pasteurizedm i l k ..............

22

Laboratory pasteurization of raw m i l k .........................

22

Coliform index..................................................

22

Enterococci index..............................................

23

Classification chart for enterococci..........................

25

Study of thermal death time.................................

26

Comparison of coliform and enterococci indexes on the oasis of
practical pasteurization as well as laboratory pasteuriza­
tion..........................................................

27

Examination of pasteurized milk for coliform and enterococci
indexes in a dairy manufacturing plant using high-tempera­
ture , short-time pasteurization and low-temperature , long­
time pasteurization for different timesand temperatures ....

27

Sources of enterococci in m i l k .................................

26

RESULTS..............................................................

29

A . Confirmation of dextrose azide-etliyl violet azide broths
for the detection of enterococci inm i l k ..................

31

h . Sources of enterococci in raw milk

32

TABLE OF CONTENTS - Continued
PAGE
C . Comparison of coliform and enterococci indexes in
pasteurized bottled m i l k ..................................

39

D. Low-temperature, long-time laboratory pasteurization......

Ll

E. Low-temperature, long-time plant pasteurization............

5>L

F . nigh-temperature , short-time plant pasteurization.........

57

G. Thermal death time of enterococci..........................

63

H. Percentage distribution of enterococci in raw m i l k ........

oo

DISCUSSION...........................................................

67

SUhj-ArtY AND CONCLUSIONS.............................................

61

LlrENATUhi. CITED ........

62

LIST OF TABLES
PAGE
Table
Table
Table
Table
Table
'I'aole
Table
Table
Table

1. Confirmation of dextrose azide-ethyl violet azide
broths for the detection of enterococci in m i l k ........

31

2. Sources of enterococci in raw milk.
Foremilk and strippings................................

33

3. Sources of enterococci in raw milk.
Milking machine.........................................

3L

L . Sources of enterococci in raw milk.
Small top milk pa i l.....................................

35

5. Sources of enterococci in raw milk.
From surface cooler followed by each cow...............

36

6. Sources of enterococci in raw milk.
Milk cans (refrigerated over night at 5.5 C ) ..........

37

7 . The logaritloriic averages of raw milk samples
(Tables 2-6 incl.) from dairy herd.....................

36

6. The comparison of coliform and enterococci indexes in
pasteurized bottled m il k ................................

LO

9 . Low-temperature, long-time laboratory pasteurization
at 61.6 C for 30 minutes................................

Lh

Table 10. Low-temperature, long-time laboratory pasteurization
at 62.5 C for 30 minutes...............................

Lb

Table 11. Low-temperature, long-time laooratory pasteurization
at o3 .L C for 30 minutes...............................

L7

Table 12. Low-temperature, long-time laboratory pasteurization
at 63.5 C for 30 minutes................................

Lt

Taole 13. Low-temperature, long-time laboratory pasteurization
at 6L.5 C for 30 minutes...............................

L9

Taole l L . Low-temperature, long-time laboratory' pasteurization
at
.O C for 30 minutes................................

50

LIST OF TABLES - Continued
PAGE
Table IS'. Low-teiTiperaturo, lonpr-time laboratory pasteurization
at 65.6 C for 30 minutes..................................

51

Table lo. Low-temperature, long-time laboratory pasteurization
at 66.2 C for 30 minutes..................................

52

Table 17. The logarithmic averages of milk samples (Tables 9-16
incl.) pasteurized at varying temperatures for a 30
minutes exposure period...................................

53

Taole l b . Low-temperature, long-time plant pasteurization at
62.0 C for 30 minutes.....................................

55

Taole 19. Low-temperature , long-time plant pasteurization at
different times and temperatures.........................

5o

Taole 2 0 . Ligh-terr.perature , short-time plant pasteurization at
72.2 C for lb seconds.....................................

56

Table 21. High-temperature, short-time plant pasteurization at
7c.o C for 1c. seconds.....................................

59

Table 22 . iiigh-temoerature , short-time plant pasteurization at
77.2 C for lo seconds.....................................

oO

Table 23. The logaritiuiiic averages 01 milk samples (Tables 20-22
incl.) p.asteurized at varying temperatures for a lo
seconds exposure period...................................

62

Taole 2a. Thermal death time of enterococci when temperature of
ol.o C was constant, ana time factorwaschanged............

oL

Taole 25. Thermal aeath time of enterococci when time of 30
minutes was constant anatemperature was changed.........

65

Taole 26. Percentage distribution of subgroups of enterococci in
milk ..........

60

LIST OF FIGURES
Fi».GE
Figure 1. Cenco-Dekliotinsky constant temperature waterbath.....

h3

Figure 2. Variation shown by different subgroups of enterococci
in litmus milk......................................

69

Figure 3. Gram stain of enterococci from dextrose aziae broth...

70

Figure 14-. Gram stain of enterococci from ethyl violet aziue
broth................................................

71

LIS! OF GRAPHS
PAGE
Graph
Graph

I. The effect of time on Streptococcus faecalis when
e x p o s e d to a temperature of txL.t C ..................

76

I I . The effect of successive increase in temperature on
the viability of Streptococcus faecalis for a 30
minutes exposure period.............................

77

Graph I I I .
Graph

The effect of time on Streptocoecus liquefaciens when
exposed to a temperature of 61.6 C ..................

78

IV. The effect of successive increase in temperature on
the viability of Streptococcus liquefaciens for a
30 minutes exposure period..........................

79

1

INTRODUCTION
There is a vast amount of confusing literature available on the
subject of enterococci.

Before Tliiercelin (1899) isolated a coccus

from human feces and used the term "enterococcus", Hirsh and Libman
(1897) classified them as Streptococcus enteritis and later Andrewes
and Border (1906) placed them under the species name of Streptococcus
faecalis.
The enterococci are markedly differentiated from the other known
species of streptococci by their combination of low minimum and high
maximum temperatures of growth and oy their greater tolerance to salt,
alkali, acid and bile.

They also differ from most streptococci in

having high thermal death points, in being resistant to relatively
concentrated solutions of methylene Dlue and in having strong reducing
action.

Tney appear in diplococcal form or in short chains and are

distinguished by their faculty of growing on ordinary media at room
temperature.
The potential pathogenicity of the enterococci, combined with their
high resistance and extreme ranges of temperature at which they grow is
of particular interest to the dairy industry.

Inasmuch as they are of

fecal origin, their appearance in milk indicates fecal contamination.
If allowed to grow extensively in milk, they give rise to taints and
abnormal flavors.

They ferment lactose, forming an acid and other ob­

jectionable by-products which make them very objectional to the fluid

2

milk industry.

For example, Streptococcus liquefaciens rapidly co­

agulates milk, then actively proteolyzes it and is sometimes responsible
for bitterness in pasteurized products .
The study of enterococci has been handicapped oy the lack of suit­
able media.

In 1950 Mallmann and Seligmann, Jr., demonstrated that

azide dextrose broth (Difco) was an excellent enrichment medium for the
growth of the enterococci and other streptococci.

The concentration of

0.02 percent sodium azide used in azide dextrose broth (Difco) inhibits
the growth of the coliform groups and allows the growth of streptococci.
Hence the azide dextrose oroth (Difco) is not only an enricliment medium
for enterococci and other streptococci, but also a selective medium for
the isolation of these organisms.

Litsky, Mallmann and Fifield (1952),

working on the development of a confirmatory medium, used O.Oh percent
concentration of sodium azide wliicli would allow St r . faecalis to grow
and yet would inhibit the gram-negative organisms. Later on they found
that O.Oh percent concentration inhibited the gram-negative bacteria but
allowed the growth of some spore-formers , such as Bacillus subtilis.
So they used another inhibitory agent, ethyl violet and thus developed
ethyl violet azide broth as a confirmatory medium for the presence of
enterococci when used with presumptive test in azide dextrose broth.
An attempt has been made to use these media for examining raw as
well as pasteurized milk samples to determine the sources of enterococci
in milk and possibly establishing an enterococci index for pasteurized
milk within the limits using Sherman classification (1937) and Bergey»s

3

manual (19U8).

The relative relationships of these organisms were

studied as well as the thermal death time of pure cultures.

u

HISTORICAL BACKGROUND
Initial Isolations.

The term enterococci was first used by Thiercelin

(1699). His findings led him to believe that enterococci are casual
agents in certain diarrhoeas, biliary infections and cases of appendi­
citis .
i-iacCullum and Hastings (1699) studied a case of acute endocarditis
and isolated a new species of organisms. They named it Kicrococcus
zymogenes and described it as follows:

"This micrococcus is very small,

occurs mainly in pairs, sometimes in short chains, gram-positive, lique­
fies gelatin slowly and is especially characterized, by its behaviour in
milk, wiiich it acidifies, coagulates and subsequently liquefies.

It

produces a milk curdling ferment ana also a proteolytic ferment, each
of which is separable from the oacterial cells.

It remains viable for

months in old cultures and is tolerably resistant to the action of heat
and antiseptics.

The micrococcus is pathogenic for mice and rabbits,

causing either abscesses or general infections."
Hirsh and Libman (1697) described S t r . enteritis as short-chained
groups and Bnglish writers, Andrewes and Horder (1906) described S t r .
faecalis as a mannite fermenter and one of the most actively saccharolytic members of the streptococcus group.
rieineman (1920) demonstrated that S tr . faecium occurs chiefly in
human feces, but also in feces from other mammals.

It ferments arabinose,

mannite and saccharose but its fermenting power is quite variable.
occurs chiefly as a diplococcus, rarely in short chains.

It

5

Dible (1921) , after studying the enterococcus and fecal strepto­
cocci, concluded that S t r . enteritis comprises the large group of true
chain-forming streptococci.
Knaysi (19^1) describes the morphology of Str. faecalis as follows:
"At the start, the cell resembles a flattened ellipsoid of revolution
which gradually evolves to become an elongated ellipsoid of revolution.
Strictly spherical cells are seldom encountered in growing culture of
Str. faecalis.
Sherman and Wing (1935) described a new species of a hemolytic
streptoccus which differed from the pathogenic species of hemolytic
streptococci in its higher maximum temperature of growth, a higher
thermal death point, and a more acid limiting pH of growth.

It also

differs from the human types in the hydrolysis of sodium hippurate and
its failure to ferment sucrose.

They suggested the name Streptococcus

hemothermophilus but later (1937) changed the name to Streptococcus
durans. The organism was thought to be closely related to the entero­
coccus group.
Sternberg (1596) isolated another streptoccus which he named Str.
septicus liquefaciens and Orla-Jensen (i92l) named a proteolytic organ­
ism S t r . liquefaciens.
Common Habitat of Enterococci.

Birge (1905) isolated from autopsied

laboratory animals a micrococcus corresponding morphologically and cul­
turally with tiiat isolated by MacCullum and Hastings (1899) with the
exception that this organism was non—pathogenic for laboratory animals.
A rennin-like ferment was secreted by the organisms.

6

ka Sg®r (1926) examined 92 strains of enterococci isolated from
cases of peritonitis and £8 strains isolated from normal intestines.
He described the enterococci as follows*

*The organism is a gram-

positive diplococcus, the individual elements being oblong or fre­
quently rhomboid and characteristically inclined to one another.
fluid media, it forms sliort chains.

In

The organism can grow at pH 5 to 10

and at temperatures between 10 and 148 C .

It is exceptionally resistant

to heat— two characteristics which differentiate it from ordinary
streptococci and pneumococci."

Gelatin was liquefied by 10 percent of

the strains.
Porch (I9itl) demonstrated that the enterococci were the predominant
group of streptococci recovered from the genito-urinary tract.
Ostrolenk ana Hunter (19h6) demonstrated that the fecal streptococci
are common in the excreta of 10 animal species and found them occurring
in significant numbers . They suggested that even though the streptococci
are outnumbered by Escherichia coli, the resistance of enterococci to
chemical agents ana possibly to other environmental factors make them of
sanitary significance as indexes of fecal contamination and pollution.
Smith (1939) studied the occurrence of S t r . zymogenes in the intes­
tines of animals and found tliat it occurred in the feces of the horse
and the cow.
Alston (1928) studied $0 strains of streptococci isolated from
the alimentary tract of man, dog and rat.
described as enterococci.

Thirty percent of them were

The organisms lie describee were the small

7

cocci, oval in shape and occurring in pairs or short chains, non­
hemolytic and capable of fermenting raannitol.
Gordon (1905) examined 20 normal stools and found at least 100,000
streptococci per gram of feces$ in the majority of the stools they ex­
ceeded 1,000,000 per gram and in some they exceeded 10,000,000 per gram.
Smith and Sherman (1936) studied the hemolytic streptococci of the
human feces and found that the commonest hemolytic streptococcus of
the human intestine is S t r . zymogenes and the second in frequency is the
related "enterococcus", S t r . durans.
Ayers and Johnson, Jr., (192U) examined 33 human fecal cultures and
found that the predominant species was Str. faecalis or enterococcus.
They concluded that S t r. faecalis is similar to, if not identical with
Str . lactis .
Donaldson (191?) stated that the French writers held the enterococcus
to be a constant inhabitant of the normal intestine, and is more commonly
found in the small intestine of infants ttiree days after birth.

He in­

dicated that on the contrary, German workers who examined the stools of
3,530 persons, found no enterococci in the stools of the people having
normal health.

He found tliat the enterococcus was pathogenic for rabbits

and probably was a variant of the S tr . faecalis group.
Oppenheim (1920) studied the human fecal streptococci and observed
that the mannite fermenting, non-hemolytic streptococci were the char­
acteristic predominant types found in the feces of normal individuals.
Hemolytic streptococci were found only occasionally in the stools of
normal, healthy people.

8

Broadhurst (1915) used the fermentative tests in indicating the
origin of a given streptococcus.

She observed tliat a large number of

mannite fermenters were from human feces and that strains from the
throats of human beings failed to ferment mannite. Mannite fermenters
were most often found in bovine mouths, milk and human feces.

Strains

found in milk were conspicuously not raffinose fermenters and rather com­
monly mannite fermenters .

But in her conclusion, she stated that these

fermentative tests did not seem to be definitely helpful in indicating
the origin of a given streptococcus.
Classification of Enterococci.

Prescott (1902) showed that the strepto­

cocci produce more acid than Bacillus coli in dextrose broth.

He also

observed that the colon bacilli appeared to be extremely sensitive to
lactic acid of certain strengths and are therefore inhibited, if not
actually killed, by the acid produced by the streptococci.

Prescott and

Baker (1908) were successful in cultivating streptococci in nearly pure
cultures by using dextrose broth of slightly greater acidity than that
produced by maximum numbers of 3. coli.
lation was 2h hours.

The incubation period for iso­

This rapid test helped to detect and separate

streptococci from other microorganisms in polluted waters.
Welch (1929) classified streptococci of human feces on the basis of
their fermentative ability and found that their fermentative character­
istics were apparently constant.

Morphology of the streptococci of the

human feces did not help in their differentiation.
Avery (1929) (1929a) differentiated hemolytic streptococci of human
and dairy origin by methylene blue tolerance and final acidity.

9

Methylene blue was bactericidal for the strains of hemolytic strepto­
cocci that fail to reduce it, but neither bacteriostatic nor bactericidal
for the strains that caused its reduction.

His work indicated that the

saprophytic strains have a greater tolerance for the methylene blue dye
than have the strains of parasitic origin.
Frobisher, Jr., and Denny (1928) working with a number of strains
of K. zymogenes demonstrated that these organisms may not belong to a
single species, since some produce alpha type, some beta type and some
gamma type hemolysis on blood agar plates . There was so much resemblence
between M. zymogenes and S t r . liquefaciens that they considered them
identical.

They emphasized that M. zymogenes should be classed as a

streptococcus and observed that there is no relation between hemolysin
and proteolytic enzyme production by these organisms.
Elser and Thomas (1936) isolated St r . zymogenes from endocarditis.
They found that all strains produce green discoloration on blood agar
plates.

Sherman, et a l . (1937) differentiated Str. zymogenes from Str.

liquefaciens , as oeing hemolytic.
zymogenes8

They observed two varieties of Str.

(l) A hemolytic but apparently non-proteolytic form, not

liquefying gelatin and causing no visible peptonization in milk.
proteolytic but non-hemolytic type.

(2) A

Hon-hemolytic strains of S t r .

zymogenes differed from S t r. faecalis , in being hemolytic.
Gibson and Malek (19Ub) observed, in their studies of the bacteriology'
of milk, tliat many strains of S tr . faecalis and its varieties are non­
hemolytic but otherwise indistinguishable from Str. durans.

10

Pearl and Harrietts (1931) examined 100 strains of fecal strepto­
cocci and 50 other strains with respect to their agglutinability by an
immune serum specific for culture of fecal streptococci and found a
definite parallelism between a serologic and bacteriophagic reactions
of the fecal streptococci studied.
Torrey and Montu (193U) studied the cultural and agglutinative re­
lationships of intestinal streptococci and demonstrated that there was
not any specific serologic type of enterococcus or diplostreptococcus
found associated with the lesions of nonspecific ulcerative colitis.
They stated tiiat M . zymogenes is a variant of the enterococcus.
Sherman and Stark (1931) stated that there were certain other
streptococci besides Streptococcus thermopiiilus which grew at h 5 C . and
above.

Sherman ana Stark (193U) differentiated S t r . lactis from S tr .

faecalis on the following basis*

Str . lactis has a lower maximum growth

temperature (Ul to U3 C.), a lower thermal death time (65 C. for 30
minutes), a lower alkaline limit for growth (less than 9.6 p H ) , and a
lower tolerance for sodium chloride.
Sherman, et a l . (1937a) studied U3U strains of St r . faecalis and
found that they all grew at 10 and h5 C .

Some cultures were able to

grow at 50 C . and all except five grew at 5 C . At least one of the
strains was able to grow at 0 C . They included Str. faecalis, Str.
zymogenes , and Str. liquefaciens in the enterococcus group.

The organisms

of the group have the faculty to cause a complete reduction of litmus in
milk before curdling.

All the cultures of St r . faecalis gave a prompt

and complete reduction of litmus in milk, with the exception of the

11

narrow zone which is exposed to the air at the surface. The property
of fermenting mannitol confirms in a broad sense but some strains failed
to ferment mannitol.
Sherman and Guns&lus (19U3) demonstrated that all members of Lancef i e l d s groups B and D tested fermented glycerol and Qunsalus and
Umbreit (19U5) showed that the Str. faecalis ferment glycerol.
Chapman (1936) , while studying the resistance of enterococci ob­
served tliat the enterococci were more resistant to sodium carbonate,
sodium bicarbonate and sodium chloride than Esch. coli, Aerobacter aerogenes. staphylococci and streptococci.

&iterococci were still viable

after contact for one hour with 3 percent sodium carbonate.

He suggested

that the interpretation of resistance to injurious agents will be simpli­
fied if enterococci are differentiated from other streptococci.
Evans and Chinn (19U7) reviewed the literature on human infections
with enterococci and found that they appear to be important casual agents
in some cases of endocarditis, intestinal disorder, abdominal infections
due to injury of the intestinal tract, infections of wounds inflicted
during war and infections of the urinary tract.

Distinguishing character'

istics of the enterococci from other streptococci were as follows t
reaction in serum of group D according to Lancefield*s precipitin test,
growth at 10 and

hB C , growth

in media containing 6.5 percent sodium

chloride, growth in media having an initial pH value of 9.6 and growth
in media containing 1*0 percent bile. The types of hemolysis and lique­
faction of gelatin were found not to be correlated with other significant
characteristics.

12

Selective M e d ia. Sc human and Farrell (I9I4I) prepared a synthetic
medium consisting of pantothenic acid, vitamin B 6 , riboflavin, glucose,
a salt mixture and 6 amino acids namely *

arginine, glutamic acid,

methionine, tryptophane, tyrosine and valine for the growth of Str.
faecalis.
Niven, Jr., and Sherman (l9Uli) studied the nutrition of the entero­
cocci and found that there was no significant difference in nutritive
requirements among strains of the four enterococcus species.
Houston and McCloy (1916) used Conradi-Drigalski's medium for iso­
lating enterococcus from p u s . This was a selective medium which in­
hibited many organisms including staphylococci. The medium turned red
in the presence of the enterococci. The heat resisting properties of
the enterococcus formed the basis of a method of further confirming organ­
isms from p u s , feces and sputum. They exposed a thick emulsion of the
coccus in broth for one and a half hours to a temperature of 55 C and
found tliat they survived.
Snyder and Lichstein (19^0) found tliat 0.01 percent concentration
of sodium azide in blood agar inhibited the growth of Bacterium proteus
and other common gram-negative forms but allowed streptococci to grow.
Mallmann, et al. (19U1) reported that slow oxidizing agents such as
potassium dichromate and sodium azide, exert a bacteriostatic effect on
gram-negative bacteria and allow the growth of gram-positive bacteria.
The gram-positive cocci, particularly the streptococci, appear to be
able to tolerate the slow oxidizing agents to a greater extent than do
the gram-positive spore bearing bacteria.

13

Kallmann and Darby (1939) In their studies on media for colifonn
organisms established a broth medium containing 1 to 5,000 concentration
of sodium aside to inhibit the coliform organisms and to allow the growth
of the streptococci.

Mallmann (191*0) using this medium suggested a new

yardstick for measuring sewage pollution.
Hajna and Perry (191*3) developed a new medium known as "S F" feedium
(Str. faecal is) for streptococci index of pollution in swimming p o o l s .
They used sodium aside in concentration of 0.05 precent as an inhibiting
agent for gram-negative bacteria and brom cresol purple as an indicator
of an acid reaction.

The medium was incubated at 1*5.5 C and an acid

reaction was thought to be almost complete evidence of the presence of
Str. faecalis.
Winter and Sandholzer (19l*6) used a modification of the White and
Sherman sodium aside penicillin medium by doubling the penicillin content
and adding 0.001 percent methylene blue.

They isolated enterococci from

raw sewage and fresh and salt water.
Mallmann and Seligmann, Jr., (1950) in a comparative study of media
for the detection of streptococci in water and sewage using lactose broth,
Mallmann's sodium azide broth (191*0) , Hajna and Perry*s S F medium (191*3) ,
azide dextrose broth (Difco) and Ritter-Treece sodium azide broth (191*8)
demonstrated that the azide dextrose broth (Difco) was the best medium
for quantative determination of streptococci.

S F broth was the poorest

medium when incubated at 1*5 C . They suggested that the macroscopic de­
termination of streptococci in azide dextrose broth, as indicated by
turbidity due to growth, may be accurate in the absence of gram—positive

rods in the test material.

In the presence of gram-negative rods the

tubes should be checked microscopically.
Jolliffe (191*8) demonstrated that the ethyl purple was an effective
bacteriostat against gram-positive organisms arxi could be successfully
used in media work.
Lit sky, Mallmann and Fifield (1952) developed ethyl violet azide
broth, a new medium for quantitative confirmation of the enterococci
from azide dextrose broth.

It was found that the enterococci were the

only group that would grow in this medium.

They demonstrated the speci­

ficity of this medium with samples from river water, sewage and soil.
A comparison of methods for the detection of enterococci showed that the
dextrose azide-ethyl violet azide broth test was the best and the easiest
of those in use at present time.
Selective Isolation of Enterococci. Mallmann (1928) used glucose and
lactose nutrient broths for quantitative measurement of B. coli and
streptococcus indexes for swimming pool water. At the end of L8 hours
incubation at 37 C . the tubes were removed and kept at room temperature
for 3 days to allow the streptococci to settle at the bottom.

The broth

was decanted carefully and a loopful of thick creamy precipitate was
stained with gentian violet.
cocci.

The smears were then examined for strepto­

He demonstrated that B. coli is not a universally reliable indi­

cator of intestinal pollution in swimming pools.

B. coli multiplies in

the swimming pool and the presence of B. coli does not necessarily
indicate pollution or danger, although the absence of B. coli is an

15

excellent Index of safety.

Streptococci are consistent Indicators of

Intestinal pollution and their numbers are directly proportional to the
number of bathers.

Streptococci do not multiply in the swimming pool

and hence their presence indicate an unsafe condition.
Mallmann and Sypien (193U) <examined natural bathing places and de­
monstrated that the streptococci index fluctuated with the bathing load.
Streptococci were not found at points where there was no bathing pollu­
tion.

The colon indexes did not respond to changes in bathing load.

The streptococci disappeared over night but the colon indexes and total
count indexes sometimes showed an increase over night.
Ardrey (1936) found in a study of the source of streptococci in
chlorinated swimming pools that the streptococci are not plentiful on
the body surfaces except where contamination from the mouth, nose and
fecal material has taken place.

Streptococci, as found in swimming

pools, were not indicators of the amount of intestinal pollution.

He

suggested that streptococci indicate the amount of pollution which has
taken place from the noses and mouths of bathers and are therefore im­
portant in determining the safety of waters in regard to transmission
of sinus and other respiratory diseases.
Ritter and Treece (19U8) studied the sanitary significance of cocci
in swimming pools and found that out of 79 strains of streptococci iso­
lated from swimming pools 65.8 percent can be classified as Str. faecalis.
They were resistant to O.U to 0.6 parts per million free available chlo­
rine for 30 minutes to one hour.

Tests for the temperature limits of

16

growth and for growth in alkaline medium were found to be moat reliable
in classifying the enterococci.
Seligman, Jr., (195l) used azide dextrose broth in the determina­
tion of the coccus index of swimming pool water. He showed the use of
the coccus group as an indicator of pollution in swimming pool water.
This group includes Str. salivarius. S t r . mitia. Str. faecalis and H .
epidermidis.
Mallmann and Litsky (1951) studied the survival of selected enteric
organisms in various types of soil and found that the enterococci are
the only organisms which could be used as the indicators of fecal con­
tamination.

They observed that the coliform organisms persist in soil

for long periods and the enterococci were found to die out rapidly.

They

suggested that the enterococci seem to be good indicators of public
health tiazard from sewage in soils and on vegetables.
Driesens (19u9) used sodium azide broth as a selective broth medium
for studying the number of streptococci in raw and treated sewage, and
longevity studies of streptococci in manured and unpolluted soils. He
demonstrated that the streptococci may be found at high population
levels in raw and treated sewage and in manured soils, and suggested for
the purposes of evaluating irrigation water and garden soils, strepto­
cocci are shown to be of greater sanitary value than are the coliforms
under certain conditions . He (1952) used the same medium to study the
number of enterococci in the feces of the chicken fed with antibiotics.
Studies of Enterococci in Dairy Products.

Kinyoun and Dieter (1912)

believed that the presence of cocci wiiich form chains in lactose bile

17

at 37 C Is presumptive evidence that milk Is contaminated with feces.
Rogers and Dahlberg (1914) studied 42 cultures from milk which
formed chains In lactose bile at 37 C , 5 cultures from infected udders,
114 cultures from bovine feces and 39 cultures from the mouths of ani­
mals .

They emphasized that the feces of the animal must be considered

as a possible source of bacteria in milk and some of them would un­
doubtedly- be found to be members of the lactic group.
Ayers and Johnson, Jr., (1914) while studying the ability- of
streptococci to survive pasteurization used 139 cultures of streptococci
isolated from bovine feces, udders, mouths, and from milk and cream.
These were heated in milk for 30 minutes under conditions similar to
pasteurization.

In the group tested 64.03 percent survived at 60 C,

33.07 percent at 62.6 C, 2.58 percent at 71.1 C and all of them were
destroyed at 73.9 C.

Using only 18 cultures from milk and cream, they

found 100 percent survived at 60 C , 9 4 .44 percent at 62.9 C , 50 percent
at 66.3 C and all of them were destroyed at 73.9 C.

They showed that

streptococci isolated from milk and cream were slightly more resistant
than those from the udder, mouth and feces.
of streptococci that survive pasteurization,

They suggested two classes
(a) Streptococci which

have a low thermal death time but because of certain resistant character­
istics peculiar to a few cells are able to survive the pasteurizing
temperatures .

(b) Streptococci which have a high thermal death time

above t.lie temperature used in pasteurization.

Heat resistance was found

to be a permanent characteristic of certain strains of streptococci.

18

Evans (1916) studied 192 samples of milk freshly drawn from normal
udders from 161 cows of 5 different dairies in 2 widely distant sections
of the country and observed that 15.1 percent were long-chained strepto­
cocci and 58.8 percent micrococci.
Davis (1916) studied streptococci commonly occurring in both pasteur­
ized and unpasteurized (certified) milk.

He found that the strains

varied among themselves, we ire more resistant to heat than human strains
of hemolytic streptococci, non-pathogenic to rabbit, acidified and co­
agulated milk rapidly and formed short or long chains, but as seen in
milk often appeared in pairs or a chain of few elements.
that these organisms may be considered to

He suggested

iiavesome sanitary significance,

Ayers and Rupp (1922) demonstrated that hippuric acid was hydrolysed
by the UU hemolytic streptococci isolated from the udders

of cows, but

not by 33 hemolytic streptococci of human origin.
Crowe (1923) used a special glucose-blood-agar medium for differen­
tiating fecal streptococci.

Blackening of the medium was found to be a

confirmatory test for fecal streptococci. He described the organism as
follows*

*A characteristic organism is that small, heat-resisting, egg-

shaped coccus, commonly known as the Str. faecalis and Str. zymogenes
is a subsection of this group of organisms,w
Brown, Frost and Shaw (1926) showed that many of the hemolytic
streptococci were harmless to man and were frequently found in the best
grades of raw and pasteurized milk.
Frost, Gumm and Thomas (1927) studied hemolytic streptococci in
certified milk on five different farms. All strains were of the bovine

19

type with the exception of four cultures which proved to be Str.
epidermicus Davis.
Long and Hammer (1936) worked with organisms imp oartant to dairy
products and found an acid-proteolytic streptococcus which was identi­
fied as Str. liquefaciens Orla-Jensen.

This organism coagulated milk by

enzyme action rather than by the formation of acid.

It remained viable

at about 5 C over a long period of time but it was killed when heated
for

hO minutes

at 65.6 C.

Niven (1938) examined 68 raw and 21*5 pasteurized milk samples for
the presence of hemolytic streptococci in milk.

He obsearved that the

prevailing types of hemolytic streptococci in raw milk were Str*. mastitidis
and the "animal pyogenes."

The most common forms in pasteurized milk

were S t r . durans and Str. zymo genes .
Gibbons (1950) using Shearman1s method for classification studied
the species of enterococci in both egg products and chicken feces.
majority of the enterococci found in the
were non-hemolytic.

egg products

and chicken

The

feces

Str. faecalis was found most frequently followed by

Str. liquefaciens, Str. durans and Str. zyroogenes . They concluded that
the enterococci were a much better index of fecal contamination in egg
p OX)ducts than the col ifoarras.
At present time, the colifoarm test of pasteurized milk is used as
an index of improper sterilization of post-pasteurization equipment or
contamination thorough defective handling or inadequate pasteurization.
Colifoanm organisms are widely distributed in the sense that there
is much opportunity for their occurrence in raw milk, in dust, field

20

water, and on utensils, grain, mixed feed, bedding, plants and so in
large numbers while their presence in raw milk may imply contamination
of a fecal nature, this is not necessarily nor generally so.
Stark and Curtis (1936, 1936a) demonstrated that the addition of
one ml. of sterile milk to lh ml. of the brilliant green bile-lactose
medium reduced the toxic action of brilliant green and allowed the
growth of the sporulating, false-test organisms, and resulted in the
growth of a larger number of the cultures which may be responsible for
false test by means of "synergism" .
It is repeatedly observed that besides coliform organisms Clostrid­
ium perfringes, Clostridium sporogenes and certain aerobic spore forming
bacilli ferment lactose.
Sherman and Wing (1933) in their study of colon bacteria in milk
concluded that the colon test is of no special value as an index to the
sanitary conditions surrounding the production of raw milk of usual
market grade.
As a matter of fact, the presence of E. coli in milk does not and
can not mean anything until and unless E. coli when found, is traced
back to its source and shown to be significant of something.

There are

so many chances that bacterial contamination will occur without the
presence of E. coli that it would seem unwise and unsafe to rely alone
upon its presence or absence as an "index to sanitation".
Taking into consideration the above facts , the coliform test does
not fulfill the necessary requirements of an index of post-pasteurization contamination in milk.

21

EXPERIMENTAL PROCEDURE
Collection of Samples
Ray Milk
Samples were drawn aseptically with 10 ml sterile pipettes from
milk cans at the Michigan State College Creamery as soon as they were
received in the plant.

Immediately after collecting they were trans­

ferred to sterile screw cap vials.
to three-fourths full.
aseptically.

Each vial was filled from two-thirds

The sample containers and closures were handled

The temperature of the sample at the time of collection

ranged from 10 - 12.8 C.

All samples were immediately cooled and stored

at about 3.3 - U.ij- C until tests were made.
Direct Microscopic Examination of Raw Milk for Total Bacterial Count
A welded loop calibrated to deliver 0.01 ml of milk was used to
transfer a loopful of the well-mixed sample to a previously heated
sterile slide.

The milk was promptly spread evenly over a square centi­

meter area using a loop, inside diameter L m m ., made of B and S guage,
no. 19 platinumiridium wire, fused on a 1 - 2 inch straight piece of
similarly alloyed wire.

The slides were dried at 10 -

C witliin an

interval of 5 minutes without cracking and peeling the milk layer on
the slide . The staining and counting of the bacteria were carried out
according to the Standard Methods for the Examination of Dairy Products
(19U8).

22

Standard Plate Count for Raw and Pasteurized Milk
The procedure recommended in the Standard Methods for the Examina­
tion of Dairy Products (191*8) was followed.

Tryptone-glucose-exfcract-'

milk agar (TGfi) wad used for measuring the total count of the raw milk
and the thermoduric count of the pasteurized milk.
Laboratory Pasteurization of Raw Milk
Exactly $ ml of each raw milk sample was transferred into 5/8 by
6 inch uniform sterile tubes.

The temperature of the sample was deter­

mined by placing a thermometer in a tube containing the same amount of
milk.

The tubes were placed into boiling water and as soon as the

temperature reached 61.8 C in the control tube, the tubes were transferred
to a water bath (61.8 C - 0.1) and were allowed to remain for intervals
of 15, 25 y «Jnd 30 minutes . At the completion of the pasteurization, the
tubes were removed from the water bath, immediately cooled and stored in
a refrigerator until they were checked for standard plate count, coli­
form index and enterococci index.
The phosphatase field test was performed according to the instruc­
tions supplied with the phosphatase field test kit.

The tablets for

the substrate and the BftC solution (2 ,2*-di-bromo quinone chlorimide)
were obtained from the Applied Research Institute, New York.
Coliform Index
The procedure recommended in the Standard Methods for the Examina­
tion of Dairy Products (19U8) was followed, using brilliant green bile

23

lactose broth.

The tubes were Incubated at 35 C for 1*8 hours. The

production of gas in the Durham *s fermentation tubes was considered to
be a positive test for the presence of coliform organisms.

The coliform

index was expressed as a most probable number of organisms per 100 ml
of milk.
Enterococci Index
This index was determined by planting suitable dilutions of the
milk in dextrose azide broth with confirmation in ethyl violet azide
broth as recommended by Litsky, Mallmann and Fifield (1952) . Three suit­
able dilutions were made and 1 ml of each dilution was transferred to
dextrose azide broth (Difco) . Five tubes of the broth were used for
each dilution.

Since the tubes contained milk, any attempt at measuring

growth by turbidity was Impossible.

Each and every positive tube from

dextrose azide broth was shaken thoroughly and U standard loopfuls
(diameter 5 mm.) of the broth were transferred to ethyl violet azide
broth.

After thorough mixing, the tubes were incubated at 35 C for 1|8

hours. The ehtyl violet azide broth used was made by the following
formulation:
Ingredients
tryptose
dextrose
sodium chloride
di-potassium hydrogen phosphate
potassium di hydrogen phosphate
ethyl violet
sodium azide
PH

Grams per liter
20

15
5
2.7
2.7
0.00120

o.U
7.0

medium sterilized at 121 C for 15 minutes

2h

In the later studies presented in this thesis on pasteurization,
the development of turbidity in the ethyl violet azide broth was con­
sidered a positive test for the presence of enterococci. The entero­
cocci index is expressed as a most probable number for 100 ml. of milk,
following the table in Standard Methods for the Examination of Dairy
Products (19^8) .
To determine if the turbidity in ethyl violet azide broth was suf­
ficient evidence of the presence of the enterococci, pure cultures were
isolated from the ethyl violet azide broth by smearing brain-heart in­
fusion agar in such a manner that discrete colonies were obtained.
After

2h hours

incubation at 35 C , U single, well isolated colonies were

transferred to brain-heart infusion broth, 6.5 percent sodium chloride
tryptose phosphate broth and litmus milk.
was incubated at

U5 C

Brain-heart infusion broth

and the other media at 35 C for H8 hours . The

litmus milk tubes were observed after

2h and

1+8 hours incubation.

The

observations were recorded as partial reduction, complete reduction,
partial coagulation, complete coagulation, proteolysis and liquefaction
of casein.
For further study, the enterococci isolated from the milk samples
were separated into the following species Str. faecalis, Str. lique­
faciens . Str. zymogenes and Str. durans. The identification followed
methods proposed by Sherman (1937) and identification according to
Bergey* s Manual (19U8), (Chart 1.).
The resistance of enterococci to 6.5 percent sodium chloride was
considered a significant testj hence, all cultures growing in sodium

25

CLASSIFICATION CHART 1
ENTEROCOCCI*

DIFFERENTIAL CHARACTERISTICS

Str. faecalis
amolysis

green

t

Str. zymogenes
Beta

1

Str. liquefaciens

Str. durans

-

Beta +

elatin liquefaction

-

+

+

-

itmus Milk
trong reduction
axLmum
emperature
or growth
orvives
0 min. at
emperature

+

+

+

-

h5 C

hS C

L5 C

5o c

62.8 C

62.6 C

62.8 C

65.6 C

annitol Acid

♦

+

+

rarely

rabinose Acid

+

+■

+

-

ucrose Acid

+

-►

+

affinose Acid

+

Hycerol Acid
Jorbitol Acid

*

rarely

+

-

+

+

-

+

+

chloride tryptose phosphate broth were further identified.

The follow*-

ing pattern was used*
sodium chloride tryptose phosphate broth
blood agar plates (horse blood)
brain-heart infusion broth

One loopful of broth from a positive sodium chloride tryptose phos­
phate broth was smeared on blood agar plates in such a way that discrete
colonies were produced.

The blood agar plates wej*e incubated at 35 C for

U8 hours at which time they were examined for hemolysis . Four isolated
selected colonies from each plate were transferred to mannitol, arabinose,
sucrose, raffinose, glycerol and sorbitol tubes for the determination of
acid production and to gelatin for the determination of liquefaction.
All tubes were incubated at 35 C for Ii8 hours.
Study of Thermal Death Time
For the thermal death time determinations, 1 ml of a

2h hour

culture

was transferred to 2$0 ml sterilized skim milk and shaken thoroughly.
Five ml of this dilution was placed in a sterile 5/8 by 6 inch test tube.
Extreme care was exercised in the delivery of the milk to the tube in
order to avoid deposition of milk on the sides of the tube. The tube was
then preheated in boiling water to a desired pasteurization temperature,
at which time the tube was transferred to the water bath— care being
exercised not to spread the milk on the sides of the tube.
pasteurization was done as usual.

The laboratory

The pasteurization was done in two ways

27

namely, (l) the temperature was kept constant and the time varied,
(2) the time was held constant and the temperature was varied.

At the

completion of each exposure, appropriate portions of the milk were trans­
ferred to tryptone-glucose-extract-milk agar plates, which were incubated
at 35 C for U8 hours to determine the number of organisms that survive
the pasteurization.
A representative culture of each of the U species of enterococci
was tested.
Comparison of Coliform and Enterococci Indexes on the Basis of Practical
Pasteurization as Well as laboratory Pasteurization.
Raw milk samples from the Michigan State College Creamery were pas­
teurized in the laboratory at 61.8 C for 30 minutes.

The coliforms and

enterococci indexes were determined on the raw milk and the pasteurized
milk.
To determine the effect of pasteurization under varied conditions,
pasteurized milk samples were collected from dairies in the environs of
Lansing and East Lansing.

Nine dairies using the low-texnperature, long­

time pasteurization (61.8 - 62 .8 C) for 30 minutes and tliree dairies us­
ing high-temperature, short-time pasteurization (71.6 - 72.2 C for 15
seconds) were checked.

Coliform and enterococci indexes were made on

all bottled milk samples obtained from these dairies.
Examination of Pasteurized Milk for Coliform and Enterococci Indexes in
a Dairy Manufacturing Plant Using both High-Temperature, Short-Time Paarteurization and Low^-Temperature , Long-Time Pasteurization for Different
Times and Temperatures
Tliis plant has 2 Majonniers cold-wall storage tanks with capacity
of 2000 and 3000 gallons.

The milk is usually held overnight at a

28

temperature varying from 3.9 - 7 .2 C . The high-temperature, short-time
pasteuriser was manufactured by Creamery Package Company.

The pasteurizer

has a capacity of 15,000 lbs. of milk per hour at regular pasteurization
(72.2 C for 16 seconds).

Two pasteurization times were used*

(l) 72.2 C

for 16 seconds for non-homogenized, (2) 77.2 C for 16 seconds for homo­
genized Vitamin D milk.
The holding pasteurizers were built by Creamery Package Company and
are commonly called multiprocess tanks. The capacity of each tank is
500 gallons.

The milk is generally held overnight and pasteurized on

the following day.
not used.

In these particular studies, the air space heater was

Samples of milk exposed to different times and temperatures

were collected.
Sources of Enterococci in Raw Milk
Milk samples were collected aseptically with 10 ml sterile pipettes
from the foremilk and strippings from 10 cows, from milking machines,
small top milk pails, coolers and milk cans.

Determinations were made

on each sample for a total bacterial count by the direct microscopic
technique and standard plate procedure. Coliform and enterococci indexes
were also determined.

29

RESULTS
The results or the Investigation are presented under the following head­
ings *
A. Confirmation of dextrose azide-ethyl violet azide broths for
the detection of enterococci in milk
B . Sources of enterococci in raw milk
1.
2.
3.
li.
5.

Foremilk and strippings
Milking machine
Small top milk pail
Surface cooler
Milk cans

C . Comparison of coliform and enterococci indexes in pasteurized
bottled milk
1. Low-temperature, long-time pasteurization at
61.8-62.8 C for 30 minutes
2. High-temperature, short-time pasteurization at
72.2 C for 16 seconds
D. Low*-temperature, long-time laboratory pasteurization at
1.
2.
3.
U.
S.
6.
7.
8.
9.

61.8 C for 30 minutes
it
ii
62.8 " n
n
n
it
63 M "
ii
63.9 " it it
n
n
n
6L.5 "
i
t
t
i
ti
65.0 "
n
n
n
65.6 "
ii
66.2 " it ii
Summary of logarithmic
counts, standard plate counts, coliform and
enterococci indexes

E. Low-temperature, long-time plant pasteurization at
1. 62.8 C for 30 minutes
2. Different times and temperatures above 62.6 C
and above 30 minutes exposure

30

F. High-temperature, short-time plant pasteurization at
1.
2.
3.
ii.

72.2 C
for 16 seconds
76.6 « » «
"
77.2 » » «
"
Summary of logarithmic averages for microscopic
counts, standard plate counts, coliform and
enterococci indexes

G. Thermal death time of enterococci when
1. Temperature of 61.8 C was constant and time
of exposure was increased from 30 minutes
upward.
2. Time of 30 minutes exposure was constant and
temperature was increased from 61.8 C upward.
H . Percentage distribution of enterococci in raw milk

31

A.

CONFIRMATION OF DEXTROSE AZIDE-ETHYL VIOLET AZIDE BROTHS
FOR THE DETECTION OF ENTEROCOCCI IN MILK

Litsky, Mallmann and Fifield (1952) reported a new test for the de­
tection of enterococci in river water, sewage and soil.

This test was

used by Ellsworth (1952) for the study of fecal streptococci in the feces
of calves fed antibiotics and by Radisson (1952) for studying enteric
streptococci in the feces of the calves fed terramycin.
Up to now, this technique has not been used to detect the enterococci
in milk.

Because of the possibility that the presence of milk might up­

set the specificity of the azide-ethyl violet combination, all positive
ethyl violet azide milk tubes were examined further to be sure that all
turbidities were due to enterococci and not to streptococci or other
bacteria that might be present.

Further confirmation was made of all

positive ethyl violet azide broth tubes from both raw and pasteurized
milk samples.
TABLE 1
CONFIRMATION OF DEXTROSE AZIDE-ETHYL VIOLET AZIDE BROTHS
FOR THE DETECTION OF ENTEROCOCCI IN MILK

No. of
Cultures

Confirmed

Percentage
Confirmation
of Samples

Percentage
Confirmation
of Cultures

103

239

yes

96.2

97.2

2

h

doubt

1.9

1.6

2

3

no

1.9

H

CVI
•

No. of
Samples

&

32

B.

SOURCES OF ENTEROCOCCI IN RAW MILK

Raw milk samples from the Michigan State College dairy herd, from
foremilk and strippings, milking machines, small top milk pails, surface
cooler, and milk cans were collected aseptically and were examined for
microscopic count, standard plate count, coliform index, and enterococci
index.

The results are presented in Tables 2, 3»

h,

5 and 6.

The logarithmic averages of raw milk samples from the above studies
are tabulated in Table 7.

The results indicate that the milk obtained

from the cow contained very few bacteria but as the milk came in contact
with milk utensils, like milking machines, small top milk pails, e t c .
the number of bacteria increased materially.
est source of contamination.

Milk cans were the great­

TABLE 2
SOURCES OF ENTEROCOCCI IN RAW MILK, FORjftUK AND STRIPPINGS

Cow No.

Microscopic Count
Foremilk
Strippings

0

1,800

0
0

0
0

6,000
6,000

7,700
2,600

300
600

20

0

1*5
0

20
0

6,000
6,000
6,000

6,000
6,000
6,000

5,000
5,200
5,000

1,100
2,500
200

5lo

li5

20
0
0

0
0
0

6,000
6,000
6,000

6,000
6,000
6,000

3,600
5,000
1,500

700
1|,000
0

0

0

0
0
0

0
0
0

6,000
6,000
6,000

6,000
6,000
6,000

8,000
15,000
6,000

500
ii,000
1,000

69

79

-

-

•

•

0
0
0

0
0
0

6,000

6,000

5,389

570

2.83

1.87

1.7

1.26

521

6,000
6,000

h90

1-PV
CM

Log.
Average

Enterococci Index
Foremilk Strippings
M.P.N.
M.P.N.

0
—

6,000
6,000

517

Coliform Index
Foremilk Strippings
M.P.N.
M.P.N.

700
1,300

6,000
6,000

523

Std. Plate Count
Foremilk Strippings

k,000

tm

-

~

-

3U

TABLE 3
SOURCES OF ENTEROCOCCI IN RAW MILK,MILKING MACHINE

Cow N o .

Microscopic
Count

Std. Plate
Count

£23

6,000

1,800

0

0

£21

6,000

300

0

US

U90

6,000

7,£00

1600

UO

£17

6,000

1,100

la

20

£2£

6,000

U ,300

1800 +

52h

6,000

hoo

6,000

i,it0U

Log.
Average

Coliform
Index
M.P.N.

0

22 +

Enterococci
Index
M.P.N.

1700
U60

£5

35

TABLE

h

SOURCES OF ENTEROCOCCI IN RAW MILK, SMALL TOP MILK PAIL

No.

Microscopic
Count

Std. Plate
Count

1

6,000

6,000

920

hB

2

6,000

U,5oo

170

18

3

6,000

3,5oo

79

93

k

6,000

600

U5

130

5

6,000

800

130

170

6

6,000

27,000

79

330

7

6,000

3,000

79

110

8

6,000

6,500

U5

hB

9

6,000

300

0

110

10

6,000

3,300

1600+

0

6,000

2,737

90+

56

Log.
Average

Coliform
Index
M .P .N.

Enterococci
Index
M.P.N.

36

TABLE 5
SOURCES OF ENTEROCOCCI IN RAW MILK FROM SURFACE COOLER FOLLOWED
BY EACH COW

Cow N o .

Microscopic
Count

Std. Plate
Count

Coliform
Index
M.P.N.

523

6,000

5,000

75

0

521

6,000

5oo

37

0

U90

6,000

800

920

130

517

6,000

10,000

1600

78

525

6,000

2,900

920

20

52U

6,000

1,100

120

78

Log.
Average

6,000

1,999

277

16

Enterococci
Index
M.P.N.

37

TABLE 6
SOURCES OK ENTEROCOCCI IN RAW MILK,MILK CANo
(REFRIGERATED OVER NIGHT AT 5.5 C)

Can No.

Microscopic
Count

Std. Plate
Count

Coliform
Index
M.P.N.

Enterococci
Index
M.P.N.

1

12,000

30,000

1600+

1*60

2

16,000

30,000

ShO

700

3

12 ,000

25,000

2hO

790

L

12,000

27,000

350

310

5

2L ,000

26,000

21+0

330

6

6,000

28,000

5^0

3,500

7

12,000

23,000

2L0

U90

6

6,000

29,000

ShO

330

9

12 ,000

hS,000

130

330

Log.
Average

11,623

26,715

363+

553

18

TABLE 7
THE LOGARITHMIC AVERAGES OF RAW MILK SAMPLES
(TABLES 2-6 INCL.) FROM DAIRY HERD

Logarithmic Average
Microscopic
Count

Std. Plate
Count

CoHform
Index
M.P.N.

Enterococci
Index
M.P .N.

Foremilk

6,000

5,369

Strippings

6,000

570

Milking Machine

6,000

1,U0U

22 +

55

Small Top
Milk Pail

6,000

2,737

90+

56

Surface Cooler

6,000

1,999

277

16

11,623

28,715

383

553

Milk Cans

2.63

1.87

1.7

1.26

39

C.

THE COMPARISON OF CQLIFOHH AND ENTEROCOCCI INDEXES
IN PASTEURIZED BOTTLED MILK

In this study, pasteurized bottled milk was picked from nine dairies
using low-temperature, long-time pasteurization and three dairies using
high-temperature, short-time pasteurization to compare the coliform and
enterococci indexes on the basis of the most probable numbers per 100 ml
milk.

The results are given in Table 8.

An examination of the results,

shows that coliform organisms were present in milk from most of the
dairies.

This might be due to inadequate pasteurization, but contami­

nation through defective handling or improper sterilization of postpasteurization equipment is more likely.

Enterococci were more suscept­

ible to high-temperature, short-time pasteurization than to low-teraperature , long-time pasteurization.

THE COMPARISON OF COLIFOHM AND iNTEROCOCCI INDEXES IN PASTEURIZED BOTTLED MILK
Indexes

t
n,•
-a 4.
,,
o
a
.
HTST Pasteurization 72.2 C
Low-Temperature , Long-Time
Pasteurization 61.8-62.8 C for 30 Kin.
seconds
1
2
lO
6
7
6
11
12
h
9
3
5
1800+

1800+

1800+

52

22

1800+

Coliform

520

1800+

52

22

0

92

Enterococci

160

1800+

22

Enterococci

Coliform
Enterococci
Coliform

1800+

1600

520

22

0

1800+

920

O

0

180

52

0

o

1600

92

22

22

52

22

o

52

52

O

0

0

160

52

52

160

520

160

52

920

0

0

180

O

O

52

600

520

o

O

52

1800+

Enterococci

920

520

180

160

1800+

520

92

o

Coliform

220

0

92

180

22

92

0

o

Enterococci

O

52

22

520

520

92

o

Coliform

0

52

220

22

52

O

220

1600

520

1600

520

22

O

22

22

220

0

O

0

1800+

220

330

0

22

161

o

o

920

0

78

520

O

52

O

180

220

0

U5

22

52

22

o

0

Enterococci
Coliform
Enterococci
Coliform
Enterococci
Coliform
Enterococci
Coliform
Enterococci
Coliform

1800+

520

1800+
0

1600
0

220

O

D.

LCW-TQtPERATURE, 10NQ-T3M* LABCKATO&r PASTEURIZATION

The percents ge-kill of enterococci by low-temperature, long-time
pasteurization was determined by laboratory pasteurization of raw milk
obtained from the Michigan State College Creamery.

The tests were made

in a Cenco-Dekhotinsky waterbath with a maximum variation in temperature
of

1 0.1

C. (Figure 1.)

The five ml. test samples of milk were placed in sterile 5/8

x. 6

in.

test tubes using care not to deposit milk on the sides of the tubes. The
tubes were then preheated in boiling water to the desired pasteurization
temperature, at which time the tube was transfered to the above mentioned
waterbath.

The tests were made at temperatures of 61.8, 62.6, 6 3 .U, 63.9,

6U.5, 65, 65.6 and 66.2 C for 30 minutes.

The number of samples tested

was:
22 samples
N
6
n
9
n
h
n
5
it
6
it
h
n
2

61.8
62.8
63 .1*
63.9
6U.5
65 .o
65.6
66.2

The results of these tests are presented in Tables 9 to 16 inclusive.
In Tables 9, 10 and 11, maximum titres of high coliform and entero­
cocci were not obtained due to the fact that dilutions used were too low.
The logarithmic averages presented for these tables on raw milk, therefore
do not present maximum titres.

They are presented, however, to show the

effectiveness, in a relative manner, of 61.6 C. in the kill of the entero­
cocci in comparison to the total count and the coliform index.

In Tables

h2

12 to 16 Inclusive, maximum titres or enterococci were obtained, so
tliat the logarithmic averages show cosiparatlve kills for each temperature.
In Table 17, the logarithmic averages of milk samples (Tables 9-16
inclusive) pasteurised at varying temperatures for a 30 minutes exposure
period have been presented.

The logarithmic averages of microscopic

count and standard plate count do not show significant differences.
Coliform organisms were completely destroyed.

As far as enterococci are

concerned, 98.72 percent of them were killed at a temperature of 61.8 C
for a 30 minutes exposure period.

Figure 1.

Cenco-Dekhotinaky constant temperature waterbath,

TABLE 9
LCW-TEMPERATURE , LONG-TIME LABORATORY PASTEURIZATION
AT 61.8 C FOR 30 MINUTES

s.
No .

Sample

Micro.
Count
(Thousand)

Std. Plate
Count
(Thousand)

Raw
Past.

1*0

50

Raw
Past.

60

Raw
Past.

35

Raw
Past.

80

5

Raw
Past.

60

6

Raw
Past.

7

Raw
Past.

1
2

3

h

8
9
10
11
12

Phosphatase
Test

Coliform
Index
M.P.N.

+

1800+
0

180,000 +
2300

1600+
0

160,000 +
1700

-

1800+
0

180,000 +
1300

+
-

1600+
0

180,000 +

80

+
-

1800+
0

180,000 +
760

65

60

+
-

1600
0

1,300
200

100

90

+
-

1800+

180,000 +
1300

+

920
0

35,ooo

220
O

28,000
0

-

55

+
-

Raw
Past.

60

Raw
Past.

75

Raw
Past.

80

Raw
Past.

90

Raw
Past.

20

1*0
90

50

+

-

70

+
—

60

+
-

70

+
-

50

+
-

o

1800+
0

Enterococci
Index
M.P.N.

L5o

1*50

il*oo
1*50

1600
0

92,000
200

1600
0

18,000 +
21*00

Continued next page

h$

ABLE 9 - Continued
Micro.
Count
(Thousand)

Std. Plate
Count
(Thousand)

Phosphatase
Test

2h

30

+

Raw
Past.

36

30

15

Raw
Past.

300

250

+

1600 +
0

18,000 +
330

16

Raw
Past.

260

200

+

1800 +
0

18,000 ♦
230

17

Raw
Past.

96

100

+

1800 +

18,000 +

o

35oo

16

Raw
Past.

36

19

Raw
Past.

20

Raw
Pas t .

bh

62

+

21

Raw
Past.

2k

36

+

920
0

U300
330

22

Raw
Past.

21

15

+

69
0

9200
HO

s.
No.

Sample

13

Raw
Past.

Hi

Log. Average Raw

Log. Average Past.

HU

61.770

Coliform
Index
M.P.N.
1600 >*•
0
1800
0

liO
110

Enterococci
Index
M .P .N.
18,000 ♦
0
230
0

1800
0

16,000 +
5UOO

1800 +
0

18,000 +
18.000 +

1800 +
0

18,000 +
330

62.867___________________1306 ♦

2U.982 +

O_________ 321

TABLE 10
LCW-TEMPERATURE , LONG-TIME LABORATORY PASTEURIZATION
AT 62.8 C FOR 30 MINUTES

S.
No.
1
2

3

Sample

Micro .
Count
(Thousand)

Std. Plate
Count
(Thousand)

Raw
Past.

60

1*0

Raw
Past.

1*8

Raw
Past.

200

Phosphatase
Test

+

w
35

+

150

Enterococci
Index
M.P .N.

180
0

2,1*00
1300

350
0

16,000
21*00

1800 +
0

3,500
0

-

1800 +
0

2,1*00
130

+
-

+

Raw
Past.

96

5

Raw
Past.

111*

110

+
-

1800 +
0

18,000 +
21*00

6

Raw
Past.

81*

82

+
-

1800 +
0

18,000 ♦
0

7

Raw
Past.

96

100

+

1800 +
0

18,000 +
1700

Raw
Past .

36

1800

18,000
270

1*

6

Log. Averi
age Raw

Log. Average Past.

30

Coliform
Index
M.P.N.

-

1*0
-

80.768

62.31

+

o

+

1100 +

8.731* +

O

161

1*7

TABLE 11
LCW-TH1PERATURE, LONG-TIME LABORATORY PASTEURIZATION
AT 63 .1* C FOR 30 MINUTES
s.
No.

Sample

Micro.
Count
(Thousand)

Std. Plate
Count
(Thousand)

1

Raw
Past.

321*

1,770

Raw
Past.

96

3

Raw
P as t .

36

1*

Raw
Past.

5

Phosphatase
Test

Coliform
Index
M.P.N.

Enterococci
Index
M.P.N.

1800 +
0

1,800,000+
0

-

1800 +
0

18,000+
950

1*0

+
-

1800 +
0

18,000-t790

111*

110

+
-

1800 +
0

18,000+
170

Raw
Past.

81*

82

+
-

1800 +
0

18,000+
0

6

Raw
Past.

2i*

36

+
-

920
0

1*,300
170

7

Raw
Past.

21

15

+
-

69
O

9,200
170

8

Raw
Past.

60

1*0

+
-

180
O

2 ,li00
1*90

9

Raw
Past.
Log. Aver­
age Raw
Log. Ave:rage Bast •

1*8

35

+

350
0

16,000
1.300

750 +

18 ,75U+

2

+
-

100

+

-

62.896

68.788

0

110

1*8

TABLE 12
LCW-TEMPERATURE, LONG-TIME LABORATORY PASTEURIZATION
AT 63.9 C FOR 30 MINUTES
Sample
s.
No. -

M ic r o.
Count
(Thousand)

Std. Plate
Count
(Thousand)

Phosphatase
Test

Coliform
Index
M.P.N.

Enterococci
Index
M.P ,N.

+
“

180
0

21*00
31*0

*•

350
0

1 6 ,0 0 0
1300

1

Raw
Past.

60

1*0

2

Raw
P as t .

1*8

35

3

Raw
P as t .

200

150

U

Raw
Past.

96

30

Log. Aver­
age Raw
Log. Aver
age Past.

6 6 .2 3 0

50.100

+

1800 +
0

3500
0

1800 +
0

21*00
0

672 +

1*,238

O

26

h9

TABLE 13
LCRf-TEMPERATURE, LONG-TIME LABORATORY PASTEURIZATION
AT 6U .5 C FOR 30 MINUTES

s.
No.

Sample

1

Raw
P as t .

2

M ic r o.
Count
(Thousand)

S t d . Plate
Count
(Thousand)

Phosphatase
Test

Coliform
Index
M.P.N.

21

15

+
—

69
0

920
U5

Raw
Past.

2h

36

+

920
0

U300
20

3

Raw
Past.

60

UO

+

180
0

2LOO
210

h

Raw
Past.

U8

35

+
*■

350
0

16,000
1300

5

Raw
Past.

l,Lho

2,310

+
•

Log. Aver­
age Raw
73 .1 2 0
Log. Aver­
age P a s t .

70.£UO

1800 +

Enterococci
Index
M.P.N.

o

1,300,000
0

373 +

11 ,U60

0

U8

TABLE lU
IXJW-TEMPERATURE, LONG-TIME LABORATORY PASTEURIZATION
AT 65 .0 C FOR 30 MINUTES
s.
No.

Sample

M i cr o .
Count
(Thousand)

Std. Plate
Count
(Thousand)

Phosphatase
Test

Coliform
Index
M.P.N.

Enterococci
Index
M.P.N.

1

Raw
Past.

30

2U

+
—

1800 +
0

230
0

2

Raw
Past.

2U

30

+
—

1800 +
0

2UOO
0

3

Raw
Past.

U8

35

+
—

350
0

1 6 ,0 0 0 .
2U00

U

Raw
Past.

60

UO

+
•“

180
0

2UOO
1300

5

Raw
Past.

200

150

+

1800 +
0

3500
0

o

Raw
Past.

96

30

+
•

1800 +
O

2U00
0

Log. Aver­
a g e Raw
58.1 j.3U
L o g . A ver­
age P a s t.

U 0 .6 8 5

933 +

O

2,372

12

51

TABLE 15
LCRf-TEMPERATURE, LONG-TIME LABORATORY PASTEURIZATION
AT 65.6 C FOR 30 MINUTES

Sample

Micro .
Count
(Thousand)

Std. Plate
Count
(Thousand)

Phosphatase
Test

1

Raw
Past.

2U

36

+

920
0

L300
78

2

Raw
Past.

21

15

+

69

9,200

0

78

Raw
Past.

30

Raw
Past.

2k

s.
No .

3

h

Log. Aver­
age Raw
Log. Aver­
age Past.

36

+
mm

2h.5kk

30

27.635

+
•

Coliform
Index
M.P.N.

1800 +

Enterococci
Index
M.P.N.

0

230
0

1600 +
0

2hOO
0

673 ♦

2,162

0

9

52

TABLE 16
LCW-TEMPERATURE, LONG-TIME LABORATORY PASTEURIZATION
AT 66.2 C FOR 30 MINUTES

S.
No.

1

2

Sample

Micro.
Count
(Thousand)

Raw
Past.

30

Raw
Past.

2k

36

Phosphatase
Test

Coliform
Index
M .P .N.

Enterococci
Index
M.P.N.

+

1800 +

230
0

o

Log. Aver­
age Raw
26.633
Log. Average
Past.

S td . Plate
Count
(Thousand)

30

32.863

+

1800 +
0

1800 +

O

2U00
0

7U 3

0

THE LOGARITHMIC AViitAGES OF i-iILK SAMPLES (TABLES 9-16 mCL.) PASTEURIZED
AT VARYING TEMPERATURES FOR A 30 MINUTES EXPOSURE PERIOD

Sample

No. of
Samples

Raw

22

Temp.
(Centi­
grade)

61.8

Logarithmic Average
Micro
Coint
(thousand)

Std, Plate
Count
(thousand)

Coliform
Index
M,P.N.

Enterococci
Index
M.P.N,

61.770

62.867

1306 +

28,982 +

0

321

1100 +

8,738 +

0

161

750 +

18,758 +

0

110

Past.
Raw

8

62.8

60.768

62.310

Past.
Raw

9

63.ii

62.896

68.788

Past.
Raw

8

63.9

86.230

50.100

0

Past.
Raw

5

6U.5

73.120

70.51*0

6

65.0

58.U3U

80.665

933 +
0

Past.

Raw

373 +
0

Past,
Raw

672 +

8

65.6

28.588

27.635

673 +

2

66.2

26.833

32.863

1800 +

98.72

98.16

99.1*2

8,236
26

99.39

11,860
88

99.58

2,372
12

99.1*9

2,162

Past._________________________________________________0___________ 9
Raw

Percentage Reduction of
Enterococci

783

99.59

vn
k*
>

5U

E.

LCW-TEMPERATURE, LONG-TIME PLANT PASTEURIZATION

Raw milk %»s pasteurized in roultiprocess tanks each having a capa­
city of 500 gallons.

It was preheated up to 62.8 C and then was held

for 30 minutes at this temperature.

The samples were examined for

microscopic count, standard plate count, phosphatase test, coliform
index and enterococci index.

Pasteurized milk samples were observed for

thermoduric count, phosphatase test, coliform index and enterococci
index.
The object of this study was to find out the behavior of coliform
and enterococci under usual plant pasteurization conditions and to com­
pare these results with the results obtained from the most accurate
laboratory pasteurization of the milk samples. The results presented
in Table 18 coincide with the results shown in Table 17.
The results tabulated in Table 19 indicate that the enterococci are
not all killed at 62.3 C. for 38 minutes, 62.3 C. for

hi minutes,

62.8

C. for 35 minutes but at 66.7 C. for 30 minutes, 66.7 C. for 68 minutes,
and when a temperature of 8h.9 C was used and the milk after reaching
the temperature, being immediately cooled, the enterococci were completely
destroyed.

TABLE 18
LOW-TEMPERATURE, LONG-TIME PUNT PASTEURIZATION AT 62.8 C FOR 30 MINUTES

s.

Sample

No.

Micro.
Count
(Thousand)

Std. Plate
Count
(Thousand)

Phospha­
tase
Test

Coliform
Index
M.P.N.

Enterococci
Index
M.P.N.

Percentage
Reduction of
Enterococci

1

Raw
Past,

1,100
i

1,150
12

+
-

1800 +
0

1,800,000 +
2,300

99.87

2

Rav
Past.

168

150
25

+

3,500,000 +
20

99.999 ♦

-

1800 +
0

Raw
Past.

156

1 ,1*30

+

1,800,000 +
330

99.98 ♦

—

1800 +
0

Raw
Past.

250

+
—

1800 +
0

920,000
130

1800 +

1,797,200

3

1*

Log. Average
Raw

Log. Average
Past.

6

291.365

300
5

521.585

9.71*0

0

99.96

211

vn

vn

LCW-TEMPERATURE, LONG-TIME PUNT PASTEURIZATION AT DIFFERENT TIMES AND TEMPERATURES

s.
No.

1

2

3

1*

5

6

7

Sample

Raw
Past.

Raw
Past.

Raw
Past.

Raw
Past.

Time
Temperature
(Minutey (Centigrade)

Micro.
Std. Plate
Count
Count
(Thousand) (Thousand)

160
38

62.3

1*1*1*
la

62.3

500
35

62.6

1000
36

Raw
Past.

30

Raw
Past.

66

Raw
Past.

h ittin g

62.8

720
66.7

288
66.7
1,1*1*0
61*.9

Phospha­ Coliform Enterococci Percentage
Reduction of
tase
Index
Index
M.P.N.
M.P.N.
Enterococci
Test

650
22

+
-

1800 +
0

1,800,000 +
1*90

99.97 +

910
3.1*

+
—

1800 +
69

1,800,000 +
1*5

99.99 +

1*50
1*

+
-

1800 +
0

1,800,000 +
110

99.99 +

900
11*

+
-

1800 +
0

1,800,000 ♦
220

99.99

600
5

+

m

1800 +
0

790,000
0

100.00

100
-

+

1800 +
0

3,500,000
0

2,310
0.5

+

1800 +
0

1,300,000
0

100.00

100.00

57

F.

HIGH-TEMPERATURE, SHORT-TIME PLANT PASTEURIZATION

A high—temperature , short-'time pasteuriser, manufactured by Creamery
Package Company, was used for this purpose.

Three different temperatures

were used, 72.2, 76.6 and 77.2 C. for 16 seconds.

The whole process was

automatic and there was no possibility of raw milk mixing with the pas­
teurized product. The number of samples tested was s
12 samples
7
"
16
»

72 .2
76.6
77.2

The results are given in Tables 20, 21 and 22 respectively.

Negative

coliform indexes and phosphatase tests were obtained at all temperatures.
The most probable number of enterococci obtained at 72.2 and 76.6 C. for
16 seconds is very low and at 77.2 C . the enterococci were completely
destroyed, except in two samples.
In Table 23, the logarithmic averages of milk samples (Tables 20-22
inclusive) pasteurized at varying temperatures for a 16 seconds exposure
period are presented.

The percentage reduction of enterococci in high-

teraperature, short-time pasteurization is more efficient than in lowtemperature , long-time pasteurization.

58

TABLE 20
HIGH-TQ1PERATURE, SHORT-TIME PLANT PASTEURIZATION
AT 72.2 C FOR 16 SECONDS
s.

Sample

Mi c ro .
Cotint
(Thousand)

Std. Plate
Count
(Thousand)

Raw
Past.

350

1,085

No.

1

Phosphatase
Test

Coliform
Index
M.P.N.

Enterococci
Index
M.P .N.

1,800,000 +
36

3k

-

1800 +
O

2

Rav
Past.

1,263

2,160
20

+
-

1800 +
20

16,100,000
U50

3

Raw
Past.

1,786

2,1*60
13

+
-

1800 +
0

9,200,000
780

1*

Raw
Past.

2,580

5,ooo
18

-

1800 +
0

2 ,300,000
0

Raw
Past.

828

1 ,1*00

+

1800 +
o

1,800,000
7 68

Raw
Past.

850

-

1800 +
0

1,600,000
330

7

Raw
Past.

696

1,1*90
31

+
-

1800 +
0

1,800,000
78

8

Raw
Past.

1*92

1,530
1*0

+
-

1800 +
0

1,600,000 +
21*00

9

Raw
Past.

1,272

2,080
15

+
-

1800 +
o

1,800,000 +
330

10

Raw
P as t .

750

700

+
-

1800 +
o

1,800,000 +

Raw
Past.

825

+

1800 +
0

1,600,000

1800 +
o

1,800,000 +
130

1600 +

2 ,501.600

5

6

11
12

io
2 ,000
20

8

336
Raw
Past.
Log. Aver­
61*3.662
age Raw
Log. Aver­
age Pa s t.

800
9

110
23
1329.100
17.753

-

+

-

+
-

2

HO
230

11*0

&

59

TABLE 21
HIGH-TEMPERATURE, SHORT-TIME PLANT PASTEURIZATION
AT 76.6 C FOR 16 SECONDS
S.

Sample

No.

6

7

Coliform
Index
M.P.N.

Enterococci
Index
M.P.N.

1,800,000 +
o

+
—

1800 +
0

Raw
Past.

1,263

2,160
10

+

—

1800 +
0

16,100,000
680

Raw
Past.

2,560

3,000
io

—

1800 +
0

16,100,000
o

1,1+90
20

•

1800 ♦
0

1,800,000 +
78

1,530
25

+
—

1800 +
0

1,800,000 +

2,080

+

1800 +
o

1,800,000 +
230

+

1800 +
0

9,200,000

1600 +

1+,21+9,700

2

5

Phosphatase
Test

1,981
23

Raw
Past.

1+

Std. Plate
Count
(Thousand)

1+1+0

1

3

Micro.
Count
(Thousand)

Raw
Past.

696

Raw
P as t .

1+92

Raw
Past.

1,272

Raw
Past.

1,768

+

9

Log. Aver­
age Raw 1,016.000
Log. Aver­
age Past.

2,1+60
11+

201+5.300

1U.651

O

230

O

22

60
TABLE 22
HICH-TEMPERATURE, SHORT-TIME PLANT PASTEURIZATION
AT 77.2 C FOR 16 SECONDS
s.
No.

Sample

Micro.
Count
(Thousand)

S t d . Plate
Count
(Thousand)

Phospha­
tase
Test

Coliform
Index
M^PiN.

Enterococci
Index
M.P.N.

lOOO

1,250
2.7

+
-

1800 ♦
0

1,800,000 +
0

Raw
Past.

200

169
5

+
-

1800 +
0

1,800,000 +
0

3

Raw
Past.

300

297
9

+
-

1800 +
o

1,800,000 +
0

U

Raw
Past.

325

3H
5

+

1800 +
0

1,600,000 +
0

Raw
Pas t .

hhO

1800 +
0

1,800,000 +
0

Raw
Past.

1,263

Raw
Past.

2,560

Raw
P as t .

1,766

Raw
Past.

10

1

Raw
Past.

2

5

1,981
23

—

+
_

1600 +
0

16,100,000
680

—

1800 +
0

16,100,000
0

2,h60
1U

+
-

1800 +
0

9,200,000

720

800
15

+
-

1600 +
0

790,000
0

Raw
Past.

552

lOOO
io

+
—

1800 +
0

9,200,000
0

11

Raw
Past.

166

150
io

+
—

1800 +
0

3,500,000
0

12

Raw
Past.

212

120
25

+
—

1800 +
0

790,000
0

13

Raw
Past.

266

100
20

+
-

1800 +
0

3,500,000
o

6

7
8
9

2,160
10
3,000
10

+
—

0

Continued next page

61

TABLE 22 - Continued

S.
No.

Sample

11*

Micro.
Count
(Thousand)

Std. Plate
Count
(Thousand)

Raw
Past.

252

300
20

-

1800 +
o

1,800,000
0

15

Raw
Past.

108

200
25

+
-

1800 +
o

1,600,000
o

16

Raw
Past.

181*

210
30

-

1800 +
0

1,800,000
1*5

Raw
Past.

253

1600 ♦
o

1,600,000
0

1800 +
0

1,600,000
o

1*66.1*00

1800 +

2 .669.1*00

12.510

0

17
ie

Raw
Past.

1,286

Log. Aver­
1*36.600
age Raw
Log. Aver­
age Past.

250
16
390
16

Phospha­
tase
Test
+

+
-

♦
-

Coliform
Index
M.P.N.

Enterococci
Index
M.P .N.

2

TABLE 23
THE LOGARITHMIC AVERAGES OF MILK SAMPLES (TABLES 20-22 INCL.) PASTEURIZED
AT VARYING TEMPERATURES FOR A 16 SECONDS EXPOSURE PERIOD

Sample

Raw

No. of
Samples

Temp.
(Centi­
grade)

12

72.2

Macro.
Count
(thousand)
663.622

Past.

Raw

Past.

1329.100
17.753

7

76.6

1,016,000

Past.

Raw

Percentage Reduction of
Logarithmic Average
Coliform Enterococci
Std. Plate
Enterococci
Count
Index
Index
(thousand
M.P.N,
M.P.N.

16

77.2

2*36,600

2065.300

1800 ♦
2

1800 ♦

16.651

0

666.600

1800 +

12.510

0

2,501,600
160

99.99 ♦

6,269,700
22

99.9999 +

2,669,600
2

99.9999 ♦

63

G.

THERMAL DEATH TIME OF EWTEROCOCCI

To study the thermal death time of four subgroups of enterococci,
namely, Str. faecalls . Str. llquefaciens. Str. zymogenes . and S tr . durans ,
pure cultures of these organisms were transfered to brain-heart Infusion
broth for 6 successive transplants and then a
for the thermal death time study.
25.

2k hour

culture was used

The results are shown in Tables

2k

and

The thermal death point was determined, first, by holding the tempera­

ture constant (61.8 C) and determining the period of exposure necessary
to obtain complete kill and second, by holding the time constant (30 min.)
and increasing the temperature until complete kill

wa.s obtained.

The

Cenco—Dekhotinsky constant temperature waterbath, with a variation of
1 0.1 C, was used for this study.

6h

TABLE

2h

THJsBMAL DEATH TIME OF ENTEROCOCCI WHEN TEMPERATURE OF
61.8 C WAS CONSTANT AND TIME FACTOR WAS CHANGED

Name of Organism

Temperature
(Centigrade)

Time
(Minute)

Str. faecalis

61.6

6U

S t r . llquefaclens

61.8

6JU

Str. zymogenes

61.8

6U

Str. durans

61.8

71

65

TABLE 25
THERMAL DEATH TIME OF QJTEROCOCCI WHEN TIME OF 30 MINUTES
WAS CONSTANT AND TEMPERATURE WAS CHANGED

Name of Organism

Time
(Minute)

Temperature
(Centigrade)

Str. faecalis

30

68.90

Str . liquefaciens

30

69MO

Str. zymogenes

30

68.90

S tr . durans

30

69.1*0

66

H.

PERCENTAGE DISTRIBUTION OF ENTEROCOCCI IN MILK

Fifty-two cultures of enterococci Isolated from milk were classified
by hemolysis on blood agar plates, liquefaction of gelatin, acid production
in different sugars and reduction of litmus milk in 2li and 1*8 hours. The
species percentage distribution of enterococci in milk is given in Table
26.
TABLE 26
PERCENTAGE DISTRIBUTION OF SUBGROUPS
OF ENTEROCOCCI IN MILK

Name of Organism

S t r . faecalis

No. of
Cultures

Percent

ill

78.8

S t r . durans

7

13.ii

S t r . liquefaciens

2

3.9

Str. zymogenes

2

3 .9

DISCUSSION
Because enterococci are common in the intestinal tracts of man and
animals, they should be good indicators of fecal contamination in foods
and w a t e r .

English bacteriologists use an enterococci test as a supple­

mentary procedure for the sanitary testing of water.
Litsky, Mallmann and Fifield (1952) reported on the enterococci test
for detecting pollution of river water, sewage and soil.

Dextrose azide

broth is employed in the test as a presumptive medium and ethyl violet
azide broth for confirmation.
The Litsky, Mallmann and Fifield procedure was used for the examina­
tion of milk, to determine the possible application of the enterococci
test in measuring the sanitary quality of raw milk, the effectiveness of
pasteurization and the detection of post-pasteurization contamination.
The cultures isolated from the ethyl violet azide broth were con­
sidered to be enterococci if they grew at

hS C

in brain-heart infusion

broth and at 35 C in 6.5 percent sodium chloride broth.

The litmus milk

reaction showed considerable variation as shown in Figure 2.

This is due

to the presence of different subgroups of enterococci in the same sample
of milk.

A gram stain of enterococci from dextrose azide broth and ethyl

violet azide broth is shown in Figures 3 and U .
A total of 2U6 cultures from 107 samples of milk were checked for
identity.

Four cultures from 2 samples were questionable and 3 cultures

from 2 other samples were definitely not enterococci.

These data show

that in this study the ethyl violet azide broth confirmative test gave an

68

accuracy of 96.2 percent for the samples examined and 97.2 percent for
the cultures examined.
The accuracy of the procedure compares favorably tilth the confirma­
tion procedures used for the detection of coliform organisms in water.
fhus for the studies presented later in this thesis, the dextrose azide
croth-et^l violet azide broth method was used as a quantitative pro­
cedure for determining the presence of enterococci in milk.
Examinations made of both foremilk and strippings showed tliat few
snterococci were present.

Only 1 out of thirteen samples of strippings

showed enterococci and then only 20 per 1O0 ml. of milk.

Coliform indexes

"Tere somewhat comparable.
The examination of milk from milking machines, small top milk pails,
coolers and cans showed higher counts depending largely upon the cleanli­
ness of the utensil and the care exercised in the operation.

In nearly

every instance the coliform count paralleled closely the enterococci index,
rhe results indicate that the enterococci test is a measurement of the
sanitary quality of milk but it is no better than the coliform test.
Inasmuch as the coliform test is already established as a sanitary index
there would be little reason even to suggest the enterococci test as a
supplementary procedure.

F ig u re 2

Variation shown by different subgroups
of enterococci in litmus milk

«

/

Figure 3

Gram stain of enterococci from -dextrose azide broth

%
Figure 1*.

dram stain of enterococci from ethyl violet aside broth.

72

To de-terrain® -the possible application or an enterococci test for
measuring the effectiveness of pasteurization, examinations of pasteurized
market milks in the local area were m a d e .

In this study pasteurized

market milk was obtained from nine dairies uwing low—temperature, long-time
pasteurization fluid three dairies using high-temperature, short-time pas­
teurization .
incidence.

Examinations were made for both coliform and enterococci

Both coliform and enterococci were found more frequently in

milk pasteurized by low-temperature, long-time pasteurization.

In general,

the incidence of enterococci was higher than that for coliforms.

The re­

sults indicate that in this particular survey post-pasteurization contami­
nation was apparently quite common because it has been well established
that coliform organisms do not survive pasteurization by either method.
The presence of enterococci in this survey does not necessarily indicate
post-pasteurization contamination.

The results show however that entero­

cocci are common to pasteurized milk and indicate the possible need of
further study to determine the effect of pasteurization on the viability
of the organisms.
Laboratory pasteurization tests were made on raw milk samples obtained
from the Michigan State College Creamery.

The laboratory pasteurization

tests were made at temperatures of 61.8 C and above for an exposure period
of 30 minutes.
cocci was 98.72.

At a temperature of 6 1 .8 C the percentage-kill of entero­
Tests made at 62.6 C gave a percentage-kill of 98.16.

Successive tests made at temperature of 6 3 .h, 63.9, 6L.5, 65.0 and 65.6 C
gave percentage-kill figures from 99.39 to 99.59.

It is interesting to

73

note that the resistant minority at all temperatures irrespective or the
initial pppulation varied from 0 to 18,000+ organisms per 100 ml. of milk.
A-n examination of these figures indicate that enterococci are apparently
quite susceptible to pasteurization temperature but in most cases a few
resistant cells existed.

This resistant minority would be sufficient to

exclude the test as a measurement of the effectiveness of pasteurization
unless the number allowable was set at some figure above the average
number observed.
In every instance, a complete kill of coliform organisms was obtained.
To study further the survival of enterococci in plant pasteurization,
one plant was selected where various pasteurization temperatures are in
current use for different milk or milks destined for further processing.
To eliminate the possibility of recontamination by post-pasteurization
which was quite evident in the first stu^y of bottled pasteurized milk,
samples were collected directly from the pasteurizer at the completion of
pasteurization.

When the milk was pasteurized at 62.6 C. for 30 minutes,

the enterococci reduction varied from 99.67 to 99.99+ percent.

The average

enterococci index before pasteurization was 1 ,797,200 and after pasteuriza­
tion of the milk was 211.

The phosphatase test and the coliform index

were both zero as would be expected.

When the temperature of pasteuriza­

tion was maintained at 66.7 C for 30 minutes or more, the enterococci were
completely killed.

These data are in agreement with the laboratory pas­

teurization t ests.
Studies were also made at the same plant using high-temperature, shorttime pasteurization.

The results obtained at 72.2 C for 16 seconds are

7h

omparable -to those obtained with low-temperature, long-time paatw rlga—
Ion at 62.6 C . When the temperature was Increased to 76.6 C Tor 16
econds, 3 out of seven samples were negative for enterococci.

W h e n the

.emperature was Increased to 77.2 C for 16 seconds only 2 out of 18 samples
;ested gave positive tests for enterococci.

The temperature necessary

Tor complete kill of enterococci In milk would appear to be a minimum
temperature of 66.7 C for 30 minutes and 77.2 C for 16 seconds.
Lear and Foster (1951) demonstrated by use of the phosphatase test
that the holding period may be a variable in different methods of pasteuri­
zation.

For example, when milk is pasteurized at 63.5 C for

30 minutes,

the percentage of total lethal heat contributed by one minute preheating
for inactivation of phosphatase is 0 .UO whereas when the milk is pasteur­
ized at 73 C, the one minute preheating contributes 29.91 percent of the
inactivation.

This is in keeping with the report by Sanders and Sager

(19U8) U. S. Public Health Service Standards provide less safety margin at
71.1 C than at 61.7 C.
If milk were pasteurized at 77.2 C a considerably greater degree of
safety would be provided and a negative enterococci test would definitely
serve as an indicator of safety.

Neither the phosphatase nor the coliform

tests would serve as both tests are negative at a temperature of 72.2 C.
Under the laboratory and plant pasteurization conditions, the complete
kill of enterococci as a group has been determined at a definite time
and temperature both in low—temperature , long-time pasteurization and In
the high-temperature , short-time process.

75

No attempt was made in these studies to determine the species of
enterococci that survived the pasteurization process.

It was felt essen­

tial to study thermal resistance of pure cultures of the four species of
enterococci under identical pasteurization conditions.
The thermal resistance of S t r . faecalls and S t r . liquefaciens is
presented in Qraphs I, II, H I , IV.
61.8 C, a kill of

99.99

faciens was obtained.

In a 30 minutes exposure period at

+ percent for both S t r . faecalls and S t r . ligue-

These results are comparable to those obtained

in the low-temperature, long-time pasteurization of m i l k . When the period
of exposure was increased, the reduction of the resistant minority was
very gradual and a few organisms (less than 100 per ml. for S t r . faecalis
and less than 250 per ml. for S t r . liquefaciens) survived up to 52 minutes,
the maximum exposure period of the t e s t .
When the temperature was increased to 6U.5 C., the kill for both
species was in excess of 99.98 + percent.

The resistant minority de­

creased very gradually when the temperature for the
was increased.

30 minutes

test period

Complete kill was not obtained until the temperature of

68.9 C for Str. faecalis and 69.U C for S t r . liquefaciens were used.
The pure culture study of thermal death time of enterococci shows
that a resistant minority occurs in each of the subgroups of pure culture
which ultimately increases the thermal death time and thermal death
temperature.
At 61.8 C the pure cultures of S t r . faecalis, S t r . liquefaciens and
S t r . zymogenes were completely destroyed in a 6h minutes exposure period
while S t r . durans was killed in 71 minutes .

In a 30 minutes exposure

Log. No. Survivors
H

M

V

J

t

'

V

A

O

'

-

^

O

D

77
feoph XX.

The tffwt of i u b o n i I ii iaortMi £n t « M M t a r i
on the viability of Str. raeealle for t 10 nlnatea

o^ow ro period

"

7

6

5

U

3

2

1

0

10

20

30

itO

50

60

Temperature In Centigrade

70

78
Cteaph 111. The «ffwt of tlao on Str. lltuofioloni nhtn
•xposod to a tanperatura of 61.B C
8

7

log. Vo. Survivors

6

5

h
© ©
3

2

1

o

10

20

30
Tina In Minutes

Uo

50

79
Gfcraph I?.

o

10

The effect of IticcMsin Increeee in ^aeperatare on
the rlafcHlty of 3tr. llquefeclene for e 30 minutes
eaqposure period •

20

30

liO

Temperature in Centigrade

50

60

70

80

period, the pure cultures of S t r . faecalis and S t r . gymogenes were killed
at 68.9 C while Str. liquefaciens and S t r . durans were destroyed at 69.ii C.
These results prove that the enterococci on the whole at pasteuriz­
ing temperature are quite susceptible to heat but a resistant minority
shows high resistance and survives at the present pasteurizing standards.
This ability of a few bacteria to withstand the pasteurizing temperature
may be due to certain resistant characteristics peculiar to a few cells.
Hen c e , the maximum time and temperature required to bring about the com­
plete destruction of enterococci is based on the resistant minorities and
not the majority of the enterococci.
The percentage distribution of subgroups of enterococci in milk
shows that the S t r . faecalis is a predominating organism wliich constitutes
7 8 . 8 percent of the total cultures of enterococci studied.

Next comes

S t r . durans and then S t r . liquefaciens and S t r . zymogenes.

These results

coincide with the statement made by Brown and Gibbons (1950) on the types
of enterococci in eggs and egg products.

m

81

SUKHAKS AND

CONCLUSIONS

The dextrose aside—ethyl violet aside broth procedure Tor the detection
or enterococci in milk was satisfactory.
Confirmation of enterococci by growth in ethyl violet azide broth
gave an accuracy of 96-97 percent.
In regular loir-temperature, long-time laboratory as well as plant
pasteurization more than 98 percent of the enterococci were killed and in
regular liigh—temperature, short-time pasteurization more than 99 percent
of the enterococci were destroyed.
The high-temperature, short-time pasteurization was found to be more
effective in destroying enterococci than was low-temperature, long-time
pasteurization.
Low-temperature, long-time laboratory pasteurization at 66.2 C for 30
minutes and high—temperature, short-time pasteurization at 77 .2 C for 16
seconds completely destroy enterococci in milk.
The thermal death time studies reveal that the majority of the entero­
cocci are susceptible to present pasteurization temperatures but a small
resistant minority survive long exposure periods .
The predominant subgroup of enterococci occurring in milk is S t r .
faecalis.
Examination of raw milk for enterococci shows that if milk is drawn
carefully, it contains very few enterococci.

But further contact with

the milking machine, milk cans, careless handling and inadequate cooling
bring tremendous increase in numbers.

82

LITERATURE CITED
Ala-ton, J . M .
1928
A n investigation of streptococci Isolated from the alimentary
tract of man on certain animals.
Jour. Bact., 16*397-1*07.
Andrewes , F . W ., and T . J . Horder .
1906
A study of
the streptococci pathogenic for man.
Lancet., 2*706-713, 775-782, 852-855.
Ardrey, W. B.
1936
The source of streptococci found in chlorinated swimming pools.
Unpublished H . S . Thesis , Michigan State College .
Avery, Roy C.
1929
Differentiation of hemolytic streptococci of human and of dairy
origin by methylene blue tolerance and final acidity.
Jour. E x p . Med., 50*1*63-1*69.
A v e r y , Roy C .
1929a
Sensitivity to methylene blue and final acidity of non-hemolytic
streptococci.
Jour. Exp. Med., 50*787-791.
Ayers, S. Henry, and W. T. Johnson, Jr.
1911*
Ability of streptococci to survive pasteurization.
Jour. Agri. Res., 2*321-330.
Ayers , S . Henry, and Philip Rupp .
1922
Differentiation of hemolytic streptococci from human and bovine
sources by the hydrolysis of sodium hippurate.
Jour. Inf. Dis., 30*366-399.
Ayers , S. Henry, and Courtland S. Mudge .
1922a
The streptococci of the bovine udder.
streptococci.
Jour. Inf. Dis., 31*1*0-50.

XV studies of the

Ayers, S. Henry, and W. T. Johnson, Jr.
1921*
Relation of S t r . faecalis to S t r . lactis.
X Studies of the streptococci.
Jour. Inf. Dis., 31**1*9-53.
Bagger, S. V.
1926
The enterococcus.
Jour. Path, and Bact., 29*225—23&.

83

Beahm, E. H.
19h2
A Bacteriological study of the streptococci Isolated from
raw retail milk.
Biol. Abet., 16*2299.
Bergey1s Manual of Determinative Bacteriology.
19Uo
The Williams and Wilkins Co., six edition.

Baltimore.

Birge, E. G.
1905
Some observations on the occurrence of H, zymogenes.
Bui. Johns Hopkins Hosp., 1 6 1309-311*
Bornstein, Siebert .
191*0
Action of penicillin on enterococci and other streptococci.
Jour. Bact., 39*383-367.
Broadhurst, Jean.

1915

Environmental studies of streptococci with special reference
to the fermentative reactions.
Jour. Inf. Dis., 17*277-330.

Brown, J. H., W. D. Frost, and Myrtle Shaw.
1926
Hemolytic streptococci of the beta type in certified milk.
Jour. Inf. Dis., 38*381-388.
Brown, Helen J., and N. E. Gibbons.
1950
Enterococci as an index of fecal contamination in egg products.
Canadian Jour. Res., 28*107-117.
Buchanan, R. E., and ELlis I. Fulmer.
1930
Physiology and Biochemistry of Bacteria.
volume II, p. 2 8 , The Williams & Wilkins C o . Baltimore.
Chapman, George H.
1936
Studies of streptococci.
IV Resistance of enterococci.
Jour. Bact., 32*hi-1*6.
Crowe , H . W.
1923
A differential medium for streptococci.
Jour. Path, and Bact., 26*51-52.
Crowe , H . W .
1933
Agglutination experiments as evidence of
nonhemolytic streptococci.
Jour. Inf. Dis., 52*192-202.

the diversity of

Davis, David John.
1916
Hemolytic streptococci found in milk; their significance and
their relation to virulent streptococci of human origin.
Jour. Inf. D i s ., 19*236-252.
Dawson, M.
1938

H . , G. L . Hobby, and Mirian Olmsteam.
V a c a t i o n in the homolytic streptococci.
Jour. Inf. Dis., 62*138-168.

Di b l e , J . H e n r y .
1921
The enterococcus and the fecal streptococci* Their
and relations.
Jour. Path, and Bact., 21**3-35.

properties

Donaldson, R.
1917
The character of the enterococcus.
B r i t . M e d . Jour., 13 *188.
Driesens, R. J.
191*9
Streptococci as indices of sewage pollution.
Unpublished M. S. Thesis, Michigan State College.
Driesens, R. J.
1952
Studies on tlie intestinal bacterial flora of thechicken.
I Studies on the effect of certain antibiotics upon
bacterial population.
II Studies on the effect of penicillin on the production
of certain 3 -vitamins.
Unpublished Ph. D. Thesis, Michigan State College, 1*8 numb,
leaves, 5 figures.
Ellsworth, Stanley Arthur.
1952
Effect of feeding antibiotics to dairy calves.
Unpublished M. S. Thesis, Michigan State College, 79 numb,
leaves, 3 figures.
Elsre , William J . , and Ruth Alida Thomas .
1936
Studies of Str. zi
Jour. Bact., 31*79Evans, Alice C .
1916
The bacteria of milk freshly drawn from normal udders.
Jour. Inf. Dis., 18*1*37-1*76.
Evans, Alice C., and Alice L. Chinn.
191*7
The enterococci* with special reference to their associ­
ation with human disease.
Jour. Bact., 51**1*95-506.

85

Frobisher, Martin, Jr., and B. Rankin Denny.
1928
A study of M . gymogenes.
Jour. Baot., 16*361-311*.
Frost, W. D., Mildred Qumm, and Robert C. Thomas.
1927
Types of hemolytic streptococci in certified
Jour. Inf. Dis., 1*0*698-705.

milk.

Fuller, C. A., and V. A. Armstrong.

1913

The differentiation of fecalstreptococci
reactions in carbohydrate media.

by their fermentative

J o u r. Inf. D i s ., 13*1*1*2-1*62.
Gibson, T ., and Y. ABD-EL-Malek.
19U6
Studies in the bacteriology of milk.
I The streptococci of milk.
II The staphylococci and micrococci of milk.
Dairy Res., 15*233-260.
Gordon, M. H.
1905
A ready method of differentiating streptococci and some
results already obtained by its application.
Lancet, 2*11*00-11*03.
Gunsalus, I. C., and W. W. Umbreit.
19l*5
The oxidation of glycerol by Str. faecalis.
Jour. Bact., 1*9*31*7-357.
H a j n a , A . A . , and C . F . P e r r y .

191*3

Comparative study of presumptive and confirmative media for
bacteria of the coliform group and for fecal streptococci.
Am. Jour. Pub. Health, 33*550-556.

Heineraan, P. G.
1920
Orla-Jensen*s classification of lactic-acid bacteria.
Jour. Dairy Sci., 3*11*3-155.
Hirsh, Jose L.
1697
I. Ein fall von Streptokokken enteritis im sauglingsalter.
Centralblatt fur B a k t ., Parasitenkunde U . Infektionskrankheiten.,
22*369-376.
Houston, Thomas, and John M. McCloy.
1916
The relation of the enterococcus to "French fever" and
allied condition.
L ancet., 191* 6 3 2 .

86

Hoskins, J. K.
19k7
Moat* probable numbers for evaluation or Coll Aero genes tests
b y reraezxtation tubs method.
Report N o . 1621. Public Health Reports United States Govt.
Printing Office, Washington, D. C .
Jolliffe, Ethel M a e .
I 9 I4.8
Studies on the effect of ethyl purple on certain bacteria.
Unpublished M. S. Thesis, Michigan State College
Kendrick^ Pearl, and Harrietts Hollon.
1931
Serologic and bacteriophagic relationships in a group of
fecal streptococci.
Jour. Bact., 21*1*9-50.
Kell in, D., and E. F. Hartree.
1931*
Inhibitors of catalase reaction.
Nature, 13l**933-93U.
Kennedy, Lura,and Harry Weiser.
1950
Some observations on bacteria isolated from milk that
grow within a psychrophilic temperature range.
Jour. Milk and Food Tech., 13*353-357 .
Kingoun, J. J., and L. V. Dieter.
1912
Bacteriological study of the milk supply of Washington,
Am. Jour. Pub. Health, 2*262-272.

D. C.

L e a r , S . A ., and H . G. Foster .
1951
Effect of preheating time on the inactivation of phosphatase
in milk.
Jour. Milk and Food Tech., ll*(l*) *131-133.
Libman, E.

1697

II Weitere mitteilungen uber die Streptokokken-enteritis bei
s&uglingen.
Centralblatt fur B a k t ., Parasitenkunde
U . Infektionskrankheiten., 22 *376-382.

Litsky, Warren, W. L. Mallmann, and C. W. Fifield.
19$2
A new medium for the detection of enterococci in water.
Submitted for publication.
Long, H. F., and B. W. Hammer.
1936
Classification of the organisms important in dairy products.
I S t r . liquefaciens.
Iowa Agri. Exp. Sta. Res. Bull., 206.

87

MacCullum, W. Q., and T. W. Hastings.

1899

A case of acute endocarditis caused by M. zymogenes
with a description of the microorganism.
Jour. Exp. Med., 1**521-531*.

Mallmann, W. L.
1928
Streptococcus as an indicator of swimming pool pollution.
Am. Jour. Pub. Health, 18*771•
Mallmann, W. L., and Adolph Sypien.
1931*
Pollution indices of natural bathing places.
Am. Jour. Pub. Health, 21**681.
Mallmann, W. L., and C. V. Darby.
1939
Studies on media for coliform organisms.
Jour. Am. Water Works Assoc., 31*689.
Mallmann, W. L.
19ii.O
A new yardstick for measuring sewage pollution.
Sewage Works Jour., 12*875.
Mallmann, W. L., W. E. Botwright, and E. S. Churchill.
191*1
The selective bacteriostatic effect of slow oxidizing
Jour. Inf. Dis., 69*215-219.

agents .

Mallmann, W. L. and E. B. Seligmann, Jr.
1950
A comparative study of media for the detection ofstreptococci
in water and sewage.
Am. Jour. Pub. Health, 1*0*286—289.
Mallmann, W. L., and Warren Litsky.
1951
Survival of selected enteric organisms
A m . Jour. Pub . Health , 1*1 *38 —1*1*.

in various types of soil.

Mallmann, W. L., Warren Litsky, and C. W. Fifield.
1952
Ethyl violet. A selective dye for the isolation of grampositive bacteria.
Stain Technology, 27*229-332.
Niven, C. F.
1938
The homolytic streptococci of milk.
The Milk Dealer, 27*61*, 66 - 6 7 .
Ostrolenk, M orris, and Albert C . Hunter.
191*6
The distribution of enteric streptococci.
Jour. Bact., 51*735.

Oppenheim, C . J.
1920
The human fecal streptococci
Jour. Inf. D i s ., 26*117-129.
Or la-Jensen. Translated by P. S. Arup.
1921
Dairy Bacteriology.
P. Blakiston's Son and Co.
Philadelphia, 1012 Walnut St. pp. 36.
Porch, Mary L.

191*1

A bacteriological study of streptococci isolated from the
genito-urinary tract.
Jour. Bact., 1*1*1*85-1*93.

Prescott, S. C.
1902
A note on methods of isolating colon bacilli.
Science N. S., 16*671-672.
Prescott, S. C., and S. K. Baker.
1901*
On some cultural relations and antagonisms of B . coli
and Houston's sewage streptococci with a method for the de­
tection and separation of these microorganisms in polluted
waters.
Jour. I n f . Dis . , 1 *196-210.
Radisson, Jean Joseph .
1952
The effect of terramycin on the performance and intestinal
flora of dairy calves.
Unpublished M. S. Thesis, Michigan State College,
132 numb, leaves, 6 figures.
Rantz, Lowell A .
191*2
The serological and biological classification of hemolytic
and nonhemolytic streptococci from human sources.
J o ur. Inf. D i s ., 71*60-68.
Ritter, Cassandra, and E. Lee Treece .
19 U 8
Sanitary significance of cocci in swimming pools .
Am. Jour. Pub. Health, 38*1532-1538.
Rogers, L. A., and A. O. Dahlberg.
1911*
The origin of some of the streptococci found in milk.
Jour. A g r i . Res . , 1 *1*91-511.
Sanders, G. P., and O. S. Sager.
191*8
Heat inactivation of milk phospliatase in dairy products.
Jour. Dairy Sci., 31*81*5-858.

Schuman, Roslyn L., And Kioh&el A. Farrell.
19Ul
A synthetic medium for the cultivation of S t r .faecalla.
Jour. Inf. Dis., 69*81-86.
Selignann, £. B., Jr.
1951
A study o f streptococci and mlcrococcl as Indicators of
pollution In swimming pool water.
Unpublished Ph. D. Thesis, Michigan State College.
Sherman, J. M., and Pauline Stark.
1931
Streptococci which grow at high temperatures.
Jour. Bact., 21-22*275-285.
Sherman, J. M., and Hellen Upton Wing.
1933
The significance of
colon bacteria in milk, with special
reference to standards.
Jour. Dairy Sci., 16*165-173.
Sherman, J . M ., and Pauline Stark.
193^4
The differentiation
of S t r . lactis from S t r . faecalis.
Jour. Dairy Sci., 1 7 *52f>-526.
Sherman, J. M., and Helen Upton Wing.
1935
A n unnoted hemolytic streptococcus
products .
Jour. Dairy Sci., 16*657-660.

associated with milk

Sherman , J . M ., Pauline Stark, and J . C . M a u e r .
1937
S t r . zymogenes.
Jour. Bact., 33*U83-U9U.
Sherman, J. M., J. C . Mauer, and Pauline Stark.
1937a
Str. faecalis.
Tour. B a c t . , 33*275-283.
Sherman , J . M .
1937b
The streptococci.
Bact. Rev., 1*1-97.
Sherman, J. M., and Helen Upton Wing.
1937c
Str. durans. N . Sp.
Jour. Dairy Sci., 20*165-167.
Sherman, J . M ., and F . R . Smith.
1938
The hemolytic streptococci of human feces.
Jour. Inf. Dis., 62*186-189.
Sherman, J . M ., and C . F . Niven, Jr .
1936a
The hemolytic streptococci of milk.
Jour. I n f . D i s . , 62 *190-201.

90

Sherman, J. M., and I. C. Gunsalus.
191*3
Fermentation of glycerol by streptococci.
Jour . B a c t ., 1*5*155-161.
Sherman, J. M., and C. F. Niven, Jr.
19l*U
Nutrition of the Enterococci.
Jour. Bact., 1*7 *335-3^*1.
Smith, Floyd R.
1939
The occurrence of Str. zymogenes in the intestines
animals.
Jour. D iry Sc i ., 22 *201-202.

of

Snyder , Marshall L . , and Herman C . Lichstein.
191*0
Sodium azide as an inhibiting substance for gram-negative
bacteria.
Jour. Inf. Dis., 67*113-115.
Sommer, Hugo H.
191*8
Market milk and related products .
second edition pp. 1*19
The Olson Publishing C o ., Milwaukee , Wis .
Spencer, L .
19i*9
Two studies of milk distribution costs and profits in the
New York Ma r k e t .
The Milk Dealer 38 (7) *50, 132, 13l*-136, 1 3 6 , ll*0-li*2.
Standard Methods for the Jfrgamination of Dairy Products.
1935
Am. Pub. ilealth Assoc., Ninth Edition.
Stark, C . N ., and L . R . Curtis .
1936
A critical study of some of the growth promoting and growthinhibiting substances present in brilliant green bile medium.
Jour. Bact., 32*375-381*.
Stark, C . N . , and L . R . Curtis .
1936a
Increased growth and gas production by Escherichia-Aerobacter
organisms in brilliant green bile medium containing sodium
formate.
Jour. Bact., 32 *385-391.
Sternberg, G. M.
1696
A text-book of bacteriology.
William Wood and Company, New York.

pp. 335-336.

Thiercelin, M. E.
1899
Sur un diplocoque saphrophyte deL*intestin
susceptible de
devernir pathogene.
Comptes rendus societe biologie. 51*269-271.

91

Tillett, William S.
193&
Fibrinolytic activity of streptococci.
Bact. Rev., 2*161-211.
Torrey, John C., and Elizabeth Montu.
193a
The cultural and agglutinative relationships of intestinal
streptococci.
Jour. Inf. Dis., 55*31*0-355.
Trout, G. M.
1953
Personal communication.
Dairy Department, Michigan State College.
Welch, Henry.
1929
Classification of the streptococci of human feces.
Jour. Bact., 17*1+13.
Winter, Charles E., and Leslie A. Sandholzer.
191+6
Isolation of enterococci from natural sources.
Jour. Bact., 51*588.