I.
IX.

Jnci lei-iCa ar,d selectivity of nedia
P c i"c -t vtc."O di z triL'V. ti on of
'd. cbls.«
"Intorai.odiatw1s " and Aerobaotei*
I I I . P r o b a b 1e source

Viv
i-i.
i q .Zj.8

'Q'JCT

of c c l i f o k h orgat .i s h s

i::

I..
II.

Ii'cid^ico arid selectivity of media.
Percentage distribution of Eschord chi?
"Inter:®Hates" and Aei-obactor
III. Probable source

by
HIGI

1i.«

A THASI3
Submitted to the Graduate School of Kichigan
State College of Agriculture and Applied
Science in partial fulfillment of the
re ciuire men ts for the degree of
SOCTOR OF PHILOSOPHY

Department of bacteriology and public Health
1948

\ p* T.— -T -. T"
•T-'v.-1 - ; T*.' m Q
i v .1
W I t ±-:Sj-i ± J 'S +. -AjI , J. O

The author extends hi s sincere gratitude to Dr*.
A. L. Bortree, Assistant He search Professor In Bacteri­
ology , for directing this investigation and his many
helpful suggestions:

so dr.. H .. J • Staf seth, Head of the

Department of Bacteriology and Public Health,

for his

valuable guidance and critic!sms in the preparation of
th i s manu sc r i r>t .

1

TABLE OF COFTLLJTS
TAODUCTIOl'T
BTO a IG/.L B AC ivG-i".CULID

o

PAA.-A.LAm PiACmDtlAS

7

Collection of Camples

7

Lew lid Ik

7

Bottled samples

8

Vat samples

8

LedI a and Reapents

9

Methods

10

Raw and bottled samples

11

Vat samples

12

Isolation of Coll form Cultures

12

Pu ri f i ea.t i on

12

G-rarn stain

13

Identification of Cultures
IRViC reactions

13
1415

Raw Rllk
Comparison of the coliform and standard
plate courts

15

15

Comparison of brilliant preen lactose bile
broth with desoxycholate ayar
17
Comparison of formate ricinoleate broth
with violet red ayar

19

P e r ce n t a ye distrib u tio n o f 1RV 1C ty p e s
an d se c t ions

19

ii

*D p. ,

Aberr ant coli

21

Probe.ble sour

oo

uri sod Ail )r

24

Incid j nc & of

24

Coooa risen of
■plate ccun ts

25

Co. ; •
■v* '<:", of
bile bro t!i Y7l
Comparison of brilliant ^reen lactose
bile broth and formate ricinoleate broth

27

Coopsrison of violet red ajar and
deso :cjcliolate agar

29

Superiority of de so >:ycho 1 a te a.jar over
brilliant green lactose bile broth

32

Percentage distribution of IHViC types
and se c ti ons

33

Comparison of the percentage distribution
of c o H f o r m suctions determined by two
methods

36

Pin point colonies

39

Vat Samples

39

Typos isolated and their percentage
distribution

41

Probable source of coliform bacteria in
pasteurised milk

41

DISCUSSION

43

SUI'JiAiiY AND CONCLUSION

52

LITERATURE CITED

56

INTRODUCTION
A general study of the coliforrn group In raw and
pasteurized milk has been made In order to elucidate the
complex coliform problem in milk.
was given to pasteurized milk.

Particular attention

Various liquid and solid

media, especially the latter, have been evaluated with a
view of obtaining the highest coliform count in the short­
est possible time..

The present standard procedures re­

quire two to three days before the results are available
and by this time the milk has been distributed..
An attempt has also been made to ascertain the per­
centage distribution of different sections or subgroups
(1) of the coliform group of bacteria, i.e.. Escherichia
coll, Aerobacter aerogenes and the "Intermediates", as
identified by the IMViC tests.

The cultures used were

Isolated from pour-plates at random, a procedure rarely
practiced by other workers*.
A new way of proving contamination after pasteuriza­
tion has been tried, but only a few samples were examined
and the results are tentative rather than final.

2

HISTORICAL BACKGROUND
The terms 'B*. coll',

'Colon*',

1Colon-aerogenes",

'E.-coll1, *Coli-aerogenes', 'Sscherichia-Aerobacter'
and ‘Colil'orm1 are all synonymously used for the
"coliform group"

(2) of bacteria by different authors at

different times*.

The Standard Methods for the Examina­

tion of' Dairy Products

(3) defines the coliform group

as "aerobic and facultative anaerobic, gram-negative,
non-sporeforming bacteria which ferment lactose with gas
formation".
Since the discovery of Escherichia coli and Aerobacter
aerogenes by Escherich in 1885, this group has been ex­
tensively studied in water and milk in Public Health Lab­
oratories.

Coliform organisms are present in practically

all raw milk and may come from barnyard manure, soil, dust.,
rarely from the udder

(4)

(5 ) and very frequently from feed

and utensils (5) .. From the public health point of view,
the coliform test in milk does not have the same signif­
icance as in water (7)«
The Milk Ordinance and Code (8), therefore, does not
provide for the use of the coliform test*.

Hovrever, the

American Association of Medical Milk Commissioners pro­
vides for the examination of certified rnilk and demands
less than ten coliform organisms per ml.-

The coliform

test as an index for judging conditions under which the
milk was produced is regarded of little significance by many

workers

(9) (10).

The Standard Kethods for the Examina­

tion of Dairy Products (11) regards the presence of both
fecal and nonfecal coliform organisms in raw and pasteur­
ized milk as direct evidence of Insanitary dairy practices.
The author fully agrees with this interpretation of the
significance of coliform organisms*.
The presence of coliform organisms in pasteurized
milk is viewed rather differently from that in raw milk.
It has been interpreted to indicate heat resistant strains,
re contamination, inadequate pasteurization, higher initial
coliform counts in the raw milk, and growth of the organisms
in the bottle.
The survival of very small numbers of coliform organisms,
especially E «. coll, in pasteurized milk, has always been
a highly controversial problem..

Survey of the literature

leaves one in doubt about the pertinence of some of the
conclusions which have been reported*.
One school of bacteriologists

(12)

(13)

(14-) (15)

(16)

observed the survival of certain strains of coliform
organisms at 143°F for 3° minutes, and reported that the
presence of coliform organisms In pasteurized milk is
neither to be interpreted as an Index of inadequate
pasteurization nor of subsequent contamination (17)••
other school of workers

(18)

(19)

The

(7) (20) stressed that

the coliform test In pasteurized milk be used as a reliable
supplementary laboratory method for the control of proper
pasteurization and plant hygiene..

Some of the Investigators

belonging to the first school did not pay any attention
to the number of cells and their physiological state when
seeding milk for laboratory pasteurization, whereas this
is Important in a heat resistance study*.

Absence of coli­

form organisms does not necessarily indicate proper pasteur­
ization or even pasteurization at all, but the presence
of typical coliform bacteria in small volumes of pasteur­
ized milk indicates some fault in pasteurization or plant
samitation.

Thus, the coliform test as an index of pasteur­

ization is applicable only to those milks which contain
coliform organisms before pasteurization (7) (21)*.
The initial concentration of coliform organisms in
raw milk does have a bearing on the survival of some cells
after pasteurization*.

Craig (22) clearly demonstrated

this point when he tested eight heat resistant strains
each of Escherichia, "Intermediate" and Aerobacter and
found that all were able to survive 143*5° F for 30 minutes
in milk when present in sufficient numbers.

No strain

tested survived when the concentration before pasteur­
ization was less than 700 coliform bacteria per ml*
Contamination after pasteurization rather than the
survival of heat resistant strains is adequately proved
by many investigators

(23)

(.24) (25)«-

Barkworth (20)

has suggested the Incubation Coliform Test (ICT) for
samples from vats or different points in the line to
control plant hygiene and locate the pockets of contam­
ination*.

Besides improper pasteurization, heat resistant strains,
high initial coliform counts in inillc before pasteurization,
and recontamination, growth of the coliform bacteria in
the bottle is an important factor which may increase the
coliform count before the milk is actually consumed..
Morris (26) stated that coliform cultures grow more rapidly
in pasteurized than in raw milk due to the bactericidal
substances present in the latter.,

Robinton (27) found

more rapid coliform growth in cream at 46.2° F than In
skim milk.

Dahlberg (28) kept pasteurized milk, having

a coliform count 0.02 per cent of the standard plate count,
at 45 to 50° F for 4 days and found that the coliform
count increased to 88 per cent of the standard plate count.
This work stresses fully the significance of growth in the
bottle..
Ever since the discovery of coliform organisms a
persistent search for new presumptive media for their de­
tection and enumeration has been in progress..

Many media

have been elaborated and discarded, being either too
inhibitory to the coliform group or giving too many false
positives.
(34) (35)

Many investigators (29)
(36)

(37)

(38) (39)

(40)

(30)

(7)

(31) (32)

(33)

(41) have studied, com­

paratively and individually, the different media and have
preferred one or the other..

Wilson recommended McConkey's

broth and agar which are the official media for the pre­
sumptive coliform test in milk in England*

In the United

States, the Standard Methods for the Examination of Dairy

Products

(11) recommends brilliant green (lactose peptone)

2 % bile broth and formate ricinoleate broth as liquid and

desoxycholate agar and violet red a^ar as solid presumptive
media for the coliform test in milk.

In the present work

the author has further studied all these media to re-valuate
them for pasteurized and raw milk,.
The classification of coliform bacteria has been very
confusing because it is a large group of closely related,
Intergrading and somewhat unstable bacteria.
(42)

Since LcConkey

(43) biochemically divided the coliform organisms into

four groups many workers

(44)

(45)

(45)

(47)

(48)

(49)

(5°)

(5 1 ) have recommended different schemes of grouping and
classification based on different biochemical reactions.
Clark and Lubs

(52) in Roger's laboratory developed the

methyl red test and Levine

(53) first emphasized the "in­

verse correlation" of the methyl red and Voges-Proskauer
tests, thus dividing Goliform organisms into Aerobacter
(V-P +, MR -) and Escherichia (V-P

MR +) sections..

For

some years it was held well-nigh perfect until Stuart and
coworkers (5^0 found that almost 10 per cent of their
cultures did not show "Inverse correlation".
Koser's

(55) citrate test resulted in the development,

and recognition of another group of coliform organisms, the
"Intermediates", which in Bergey's Manual of Determinative
Bacteriology (56) is termed Escherichia freundll.
and Levine

(57) and Vaughn and Levine

Mitchell

((58), by using nucleic

acid and its degradation products as the source of nitrogen

for coliform organisms, have reported additional evidence
of the "Intermediates" as a separate section and have added
a new species, Escherichia intermedium, besides E- freundll,
to this section.
Parr (59)

(6 0 ) (1) analyzed the data presented in

papers published from 1924 to 1937 and suggested that the
ILTViG quartet of tests, being most frequently used , should
in the future be employed for coliform classification.
Stuart and coworkers

(54) incorporated cellobiose with

the ILTV10 tests..
The author agrees with Parr's classification (1) and
has followed it in the present work to determine the per­
centage distribution of E . coll, A. aerogenes and "Intermedi­
ate" ’sections of the coliform group of bacteria in milk.
The section, E. ooli, is comprised of three IEViC types,
++— , -+—
types,

and +---, the section,

"Intermediate", of ten

-++-, +-++, ++-+,

+++*, -+++,

+++- and +— +, and the section, A. aerogenes, of three
types, — ++, — +- and --- +,.
EXPERIMENTAL PROCEDURES
Collection of Samples
Raw milk
Samples were drawn aseptically with a 10 ml sterile
pipette from cans at the i-i.S.C. dairy barn one hour after
milking..

The temperature of samples at the time of col­

lection ranged from 50 to 53° F*.

Samples were brought to

the laboratory in ice-cooled containers and tested imrnedi-

8
ately.

These samples represented a herd of 60 cows.

The other samples of raw milk from each farmer were
collected at the M.S.G. creamery in the forenoon..

These

samples were aseptlcally drawn from the weighing pan hold­
ing milk of one farmer at a time..

The samples collected

were from 48 individual producers.
Bottled samples
One of the very first few bottles from a vat was col­
lected from the M.S.G. creamery v/ithin 3 hours of pasteur­
ization.

These bottles were kept below 40° F in mechanically

refrigerated rooms..
Bottles were also obtained from individual vats from
four other creameries; namely, Lansing Farm Products,
Heatherwood Farms, Arctic Dairy, and Lansing Dairy..

These

bottles were collected in November and December within 4
to 6 hours after pasteurization..

No ice-cooled containers

were used as the atmospheric temperature was below 40° F
and the samples were tested immediately..
Vat samples
Four ounce bottles, the neck and stopper of each
connected with a long string, were wrapped in paper and
sterilized in the oven so that when samples were drawn
from the vat no contamination could occur due to the
bottles.

These bottles were used in duplicate for samples

from each vat..

Immediately after pasteurization (pasteur­

ization temperature and time ranging from 143 to 146° F
for 30 to 35 minutes) the bottles,

suspended by the string,

9

were dipped into the pasteurized milk and withdrawn.

They

were stoppered, the outside sanitized by 200 pprn chlorine
solution, and cooled to 98° F at which temperature one
bottle of each sample was Incubated.
Media and Reagents
The following dehydrated culture media from Difco
Laboratories

(61) were used and the directions of the

manufacturers strictly followed.
1.

Bacto-Brilliant Green Lactose Bile Broth

2.

Bacto-Formate Ricinoleate Broth (71)

3.

Bacto-Violet Red Agar

4.

Bacto-Lactose Broth

5„

Bacto-Tryptone Glucose Extx’
-act Agar (to which had
been added 1 f0 sterile skim milk)

6.

Levine Eosin Methylene Blue Agar

7.

Bacto-M,R.-V.P. Medium

8.

Bacto—Koser Citrate Medium

9.

Bacto-Tryptose Agar-

1 0 . -Bacto-Tryptone
Dehydrated desoxycholate agar (62), prepared by the
Baltimore Biological Laboratories, was dissolved by heat­
ing to the boiling point,

then dispensed in 10 to 15 ml

quantities in sterile test tubes, sterilized in flowing
steam for 30 minutes

(11), and stored.

Before use, the

tubes were just melted by heating 3 to 4 minutes in the
Arnold steamer.
Lauryl sulphate tryptose broth (63) was prepared as

10
described in the Standard Methods for the Examination of
Water and Sewage (64).
The phosphs.tase (field)

pest was performed according

to the instructions supplied with the Phosphatase Field
Test Kit.

The tablets for the substrate and the BQC solu-

tion (2,6-dibromoquinone chlorimide) were obtained from
the Applied Research Institute, New York.
The reagents for the Gram stain were prepared accord­
ing to Hucker1s modification described in the Manual of
Methods for Pure Culture Study of Bacteria-, Leaflet IV
(65).
For the indol test reagent five grams of cp..paradimethyl aininobenzaldehyde was dissolved in 75
alcohol to which 25

ml of concentrated HG1

reagent should have

a yellow color.,

For the methyl

red indicator solution

amyl

was

added..The

0.1

gram of

methyl red was dissolved in 300 nil of 95 per cent ethyl
alcohol and diluted to 500 ml with distilled water.
For the Voges-Froskauer test reagents a 5 pe** cent
alphanaphthol solution was made in absolute ethyl alcohol
and a 40 per cent KOH solution in distilled water..
Me thods
The procedures recommended In the Standard Methods for
the Examination of Dairy Products

(3) were followed in

diluting samples and Inoculating tubes and plates.Two solid
media, desoxycholate agar and violet red agar, and three
liquid media, brilliant green lactose bile broth, formate-

11

ricinoleate broth, and lauryl sulphate tryptose broth were
employed for the coliform count and tryptone glucose ex­
tract milk agar for the standard plate count..

Controls

were used for each medium and technique.
Raw and bottled samples
Raw milk dilutions, ranging from 1:10 to 1:10,000,
in geometric series, were planted in broths

(5 tubes for

each dilution) and from 1 to 1:100 in solid media in the
summer season, while dilutions from 1 to 1:1,000 in
broth and 1 to 1:10 in solid media were used in fall and
winter.. For the standard plate count dilutions 1:100,
1:1,000 and 1:10,000 were used., Pasteurised milk was
used in portions of 1 ml and 0.1 ml for broths, 1 ml and
2.5 ml for solid media, and dilutions of 1:100 and 1:1,000
for the standard plate count.
To prevent the formation of atypical coliform colonies,
violet red agar and desoxycholate agar, after inoculation
and solidification, were covered with 3 to 4 ml of the
respective sterile agar..

Standard plates and broth tubes

were incubated at 35 to 37° C fob 48 hours while desoxy­
cholate and violet red plates were Incubated at 35 to 37° 0
for 20 to 24 hours.
Standard plate colonies were counted with the Quebec
counter..

Typical dark red colonies, at least 0.5 mm in

diameter, were counted on the desoxycholate and violet
red plates..

Hoskin's table given in the Standard Methods

for the Examination of Dairy Products

(3) was used to

12

compute the "EPN"

(Most Probable Number) in the broth tubes.

Those broth tubes having less than 10 per cent gas were
confirmed on eosin methylene blue agar and then the "MPN"
was determined.
Vat samples
One of the duplicate bottles was used for the phos­
phatase field test (66).

Each of 5 brilliant green

lactose bile broth tubes was planted with 1 ml of milk
for the initial coliform count and tryptone glucose e x ­
tract milk agar plates with 1:100 and 1:1,000 were poured
for the initial standard plate count.
The other bottle,

containing about 100 ml of milk,

was incubated at 35° G for 6 to 8 hours and then 4 plates
of desoxycholate agar were seeded each with 2.5 ml of milk
and 3 plates with 1 ml, 0.1 ml and O.JOl ml of milk*. These
plates were incubated at 37° 0 for 18 to 24 hours, the
positive plates being used for purified culture isolation
and typing as described b e l o w . ,
Isolation of Coliform Cultures
Purification
From presumptive positive desoxycholate and violet
red plates having less than 10 typical deep red colonies,
each colony was picked and transferred to individual
brilliant green lactose bile broth tubes whereas from
plates having a larger number of such colonies, 4 to 6
representative colonies were picked at random and trans­
ferred to individual brilliant green lactose bile broth

tubes..

The inoculated tubes were incubated at 37° C for

24 hours and a trace of each liquid culture was streaked
with a bent needle on solid eosin methylene blue plates,
which were incubated at 37° C for 24 hours..

A discrete

colony from each plate was picked and transferred to an
individual lactose broth tube which was incubated at 37° 0
and immediately after the appearance of gas was restreaked
on eosin methylene blue agar plates as above.

From each

of these plates a discrete colony was picked and streaked
on a tryptose agar slant and incubated for 24 hours at
37° C.

This was the procedure followed throughout for

the isolation and purification of cultures.

These cultures

were next subjected to gram staining for the completed
coliform test.
Gram stain
Hucker’s modification of the Gram stain was followed
and the smears were stained as described in Leaflet IV
of the Manual of Methods for Pure Culture Study (.65)
The cultures which were gram-negative, non-sporef orraing
rods were next typed.
Identification of Cultures
The IMV1C reactions were utilized for the identi­
fication of coliform organisms (purified as described
above) and 24 hour slope cultures were used.

A trace of

the culture was transferred with a needle into a tryptone
broth tube, the needle was shaken a few times, and the
same needle was next used to seed a M.R.-V.P. broth tube.

14

Then the needle was sterilized, cooled, and a trace of
the same culture was used to seed two tubes, one of
citrate and the other of M.R*-V.P. broth, care being
taken to inoculate the citrate medium with very light
inoculum so as not to carry any nutritive matter into the
broth.

The methyl red and Voges-Proskauer tubes were in­

cubated at 30° C for 5 hays and 24 to 48 hours respective­
ly (67)

(64).

The tryptone broth and citrate tubes were

incubated at 35 to 37° 0 for 24 hours and 72 hours re­
spectively .
IMV1C reactions;
1.

Indol differential test.

To 5 ail of 24-hour

tryptone broth coliform culture was added 0.2 to 0.3 ml
of amyl alcohol indol reagent and shaken.

A dark red

color, developing on the surface within 10 minutes, con­
stituted a positive test, the original color of the indol
reagent a negative test, and the intermediate a question­
able o n e .
2.

Methyl red differential test.

To 5 -dl of a 5

day old k.R.-V.P. broth culture 5 drops of methyl red in­
dicator solution were added.

A distinct red color was

recorded as positive and a distinct yellow color as neg­
ative .
3.

Voges-Proskauer differential test.

About 0,6 ml

of 5 peF cent alphanaphthol in absolute alcohol and 0.2 ml
of 40 per cent KOH solution were added to 1 ml of 24 to
48 hour M.R..-V.P,. broth culture and shaken until the white

15

precipitate just dissolved.

The development of a crimson

to ruby color within 2 to 4 hours was reported as positive
and no color formation as negative.
4.

Sodium citrate differential test.

Koser's sodium

citrate broth tubes were observed for growth after 72 to
96 hours incubation at 37° 0.

Visible growth was reported

as positive and no growth as negative.
The results of these differential tests for each
culture were recorded in the sequence of Indol, Methyl red,
Voges-Proskauer, and Citrate test,
IMViC tests.

together known as the

All coliform cultures thus were divided into

three subgroups or sections: E«. c o l i , "Intermediate", and
A. aerogenes.
RESULTS
Raw Milk
Coliform and standard plate counts were determined
for each of the 48 producers'

milk supplied to the II.S.C.

creamery during August, 1946.

Fifty-six samples (16 from

I-l.S.C. creamery producers and 40 from the M.S.C. dairy
barn) were studied thoroughly in January, February, March,
August, September and October, 1947.

As the number of

samples was not large, the results presented are only
tentative.
Comparison of the coliform and standard plate counts
Brilliant green lactose bile broth, formate ricinoleate
broth, desoxycholate agar and violet red agar were used
for the coliform count whereas tryptone glucose extract

16

inilit agar was utilized for the standard plate count.
Lauryl sulphate tryptose broth (6 3 ) proved unsatisfact­
ory (68) when inoculated with 1 ml milk and hence was
rejected in further work..

In higher dilutions it gave

fairly comparable results.
The samples from the M.S.G. producers had very high
coliform and standard plate counts.

The M.S.G. dairy

barn samples during the same se;-3on gave a much lower
Incidence of coliform and standard plate counts.
A general correlation was found between the c o n ­
form and the standard olate counts as shown in Graph I.
To avoid plotting 88 samples on the graph, the arithmetic
averages of the coliform and standard plate counts of
about every 4 samples, the counts of which were in a
certain range (i.e., coliform count from 100 to 1,000 and
standard plate count 10,000 to 100,000), were plotted.
This curve clearly shows that a higher coliform count is
concurrent with a higher standard plate count and vice
versa.

The lower incidence of coliform and standard plate

counts in M.S.G. dairy barn samples was due to clean pro­
duction and lower keeping temperature of the milk.
The seasonal variations in coliform and standard
plate counts in milk from the same producers, presented
in table 1, show how sharply the coliform count falls in
winter below that of the summer..

IlQ-LLL

Log of
Count

Standard Plate Count

Collform Count

M . 3 . 0 . Dairy
Barn Samples

1 *2

Kumber of 5amules

21..

17

TABLE 1
Seasonal Variation of Coliform and Standard Plate Counts

Coliform Count per ml

S.. P. Count per ml

Produc­
er M o .
Summer

Winter

Summer

1

18,000

o
o
uon
h~>

Winter

810,000

100,000

2

2,400

900

30,000

65,000

5

2,800

12

135,000

63,000

8

79

0.5

176,000

8,000

9

4,900

4

236,000

17,000

13

1,600

4.5

172,000

1,100

18

150

2

90,000

20,000

30

600

8

20,000

4,800

59

6,350

130

558,000

81,000

61

79

5

250,000

8,000

Comparison of brilliant green lactose bile broth with
desoxycholate agar
In comparing the various media for coliform counts
one medium was arbitrarily selected as standard, the count
by that medium was assigned a value of 100 per cent, and
the counts on other media were compared for calculating
efficiency.

Regarding brilliant green lactose bile broth

as a standard, desoxycholate agar was compared and its per
cent efficiency calculated.

Desoxycholate agar gave higher

coliform counts in 25 samales and brilliant green 1actose

18

"bile broth in 22 samples while in 5 samples the counts
were equal.

Arithmetic coverage of the total coliform

count of all samples of brilliant green lactose bile
broth was higher than such an average of desoxycholate
agar.

If brilliant green lactose bile broth was ICO per

cent efficient, desoxycholate agar proved 95*5 per cent
efficient.

The two media were very closely comparable

as shown in table 2 .
rn » -OT T -.

'1 'A D J . a i i

Comparison of Standard Presumptive Liquid Media with Solid
Media in Milk
Number of Samples
Media
Total Higher Equal

Brilliant
green lactose
bile broth

22

Average
Coliform
Count
pe r ml

Per cent
Efficiency

73.45

100

5

53
Desoxycholate
agar

26

70.13

95.5

Formate
ricinoleate
broth

6

212.8

100

333.6

160

30
Violet red
agar

.

24

0

19

Comparison of formate ricinoleate broth with violet red ap;ar
Thirty samples of milk from r-i.S.G. creamery producers
were tested and the coliform counts obtained by formate
ricinoleate broth and violet red agar were compared exact­
ly a.s above.

When formate ricinoleate was regarded 100

per1 cent efficient, violet red agar was found 160 per cent
efficient as shown in table 2 ,.
From violet red plates representing 16 samples, 154
typical deep red colonies were subjected to the completed
coliform test arid it was revealed that only 85*2 per cent
proved coliform positive while under similar experimental
conditions desoxycholate agar gave 96 per cent coliform
positive colonies.

Hence, the higher coliform count ob­

tained with violet red agar may be due to a higher number
of false positives.
Percentage distribution of Ii-iVlG types and sections
Fifty-five samples were subjected to IHViC reactions.
About 10 colonies, either from a desoxycholate or violet
red plate, representative of a single sample were picked
at random.

Completed coliform tests were performed on

55° such colony cultures which had first been purified.
Out of 16 possible IhViC types, 13 were identified.

The

average percentage distribution of these types end their
per cent occurrence are given in table 3*

For percentage

distribution of sections as E«. c o l l , "Intermediate", and
A. aerogenes.

Parr's

(1) grouping of types is followed.

It has been found that the oercentare distribution of these

20

TABLE 3
Average Percentage Distribution of Various Types and
Sections of Coliform Group Organisms Determined in 55
Hi Ik Samples by IllViO Reactions

Cul­
tures
-Typed

Con­
form
Section

Per
IHViC cent
Types Occur­
ence
T +-

558

54

% NonSection inversely
Type %
Correlated
<

20..27
CO

13

3.25

- +- +

30

12.24

—+ + ”

10

3.38

3.38

+ ++ +

6

2.13

2.13

++-+
"Intermediate" “ + ++

4

1.90

8

0.7 0

0.70

----

6

o.6 o

0.60

+--+

c

o
.
ui

XL... UUli

0*55

■f— + +

2

0.18

-+ +

85

47.46

---+

25

4.39

13

2.95

A.aerogenes

2 10.68

100,.00

Total ----- - - - - .

...

54.80

4.-39

100.00

11*75

21

three sections varies, with the locality (69) and. condi­
tions of production at the farm and the handling and
hauling of the milk..

The average percentage distri­

bution in 16 samples from M.S.G. creamery farmers was
E . col l, 4? per cent; A.- aero genes, 12 per cent; and
"Intermediate", 41 per cent while 39 samples from the
dairy barn included E . coll. 16 per cent; A. aerogenes, 79 per cent; and "Intermediate", 11 per cent.
These figures illustrate the previous statement..
Of the 13 types obtained, 6 showed no "inverse
correlation"

(53) of methyl red and Voges-Proskauer tests.

These types represent about 12 per cent of the total type
percentage.
Aberrant collform organisms
The term "aberrant" has been used for the "slow
lactose fermenters" as suggested by Stuart and coworkers
(70) and is comprised of 4 types:
1.

Mlcroaerogenic coliform bacteria

2.

Papilla-forming coliform bacteria

3.

Pseudoaerogenic coliform bacteria

4.

Anaerogenic coliform bacteria

An examination of 40 samples showed 18 samples
positive for aberrant coliform bacteria.,

Their average

percentage, determined from 40 samples, was 13 and
composed of 47 out of 38S cultures.
and anaerogenic types were Isolated.

Only mlcroaerogenic
The former, 4.5 per

cent (15 cultures) and the latter, 8.5 per cent

22

(32 cultuz-es).

None of these aberrant forms belonged to

the E . coll section on the basis of Parr's Il-iViC reactions.
Among the anaerogenic coliform bacteria the"Intermed­
iate" type having the Ii-rViC formula - + + - was the most pre­
valent.

Three chromogenlc (yellow) cultures, with the

IMV1G formula -■++-, were isolated from one sample.

Yale

(3 2 ) also found yellow cultures in his s.berrant strains
and classified them as Flavobacterlurn.

Thirty anaerogenic

cultures produced acid but no gas in lactose broth while
two cultures produced neither acid nor gas.

All these

cultures were gram-negative, non-sporogenous rods, giving
typical deep red colonies on desoxycholate or violet red
agar.

Collform-like surface colonies, some with metallic

sheen, appeared on eosin methylene blue plates..
The mlcroaerogenic cultures produced a bubble of gas
in lactose broth in 3 to 9 days and were white to pink on
eosin methylene blue plates after 2 b hours.

Dark centers

gradually developed concurrently with the development of
gas in lactose broth tubes..

These cultures belonged most­

ly to A . .aerogenes. a few to "Intermediate", but none to
E.. coll.
Probable source of contamination
No definite source of contamina.tion was ascertained*
Aseptically drawn milk from 11 cows gave negative coliform
results when 10 ml quantities were tested and all cows
were free from any udder infection.

Rinse water from cans,

milking machines, pails, and swabs from the surface cooler

23

and milk tank were tested for coliform bacteria and proved
negative except for one milking machine, having a 1.6
coliform count per ml of 500 ml rinse water,

milk samples

were collected the same day from cans soon after milking
and cooling, and were then tested immediately so as not
to give any chance for growth of bacteria.

Forty to 130

coliform bacteria per ml were found which on typing proved
to be A. aeropenes or "Intermediate" but not E. coli, inW M

M l

■-

.iW

»

■

dicating non-fecal contamination •

'

' ■ I' ^

hone of the 40 salaries

gave more than 50 per cent E. coll and the average distri­
bution was only 16 per cent.
On the other h a n d , of 16 samples from M ,3 .C . creamery
producers, 7 samples showed 67 to 100 per cent E. coli in
January and February as shown in table 4.
TABLE 4
Predominant Distribution of E. coll in Certain Producers*
Samples
Standard
Plate
Count

Producer
Number

Coliform
Count
per ml

2

80

8,200

100

8

9

c
>;

17,000

83

6

61

4

8,000

100

4

18

2

11,000

67

3

8

0..5

8,500

100

4

30

6

4,800

92

12

59

130

31,000

100

10

% E.

coll
(++--)

Cultures
Typed

24

However, these coliform and standard plate counts were
not high but the overwhelming orevalence of E. coll in­
dicated fecal contamination.
Pasteurized Milk
One hundred and forty-seven bottled samples from 5
creameries were studied exhaustively during January,
February, October, November and December, 1947.

No ice

containers were used due to the lower atmospheric temp­
erature.

Two standard liquid media, brilliant green

lactose bile broth and formate ricinoleate broth, and two
solid media, desoxycholate agar and violet red agar, re­
commended by the Standard methods for the Examination of
Dairy Products

(7) were evaluated.

Twenty to twenty-two

ml (about 5 nl in each medium) of milk from each sample
were tested for coliform organisms.

Of 147 samples, 96

(about 65 per cent) proved positive by one or more of the
media.

This higher percentage of positives is partly due

to the recovery of coliform organisms by different media
and partly due to the larger quantities of milk tested.
In some cases a small amount of growth of coliform bacteria
in the bottle may have occurred.
The coliform count in positive samples, being very
low as compared to that of raw milk, was determined per
100 ml.
Incidence of coliform organisms
The samples tested from Lansing creameries were some­
times 6 to 8 hours old and slight growth might have taken

25

place during this interval

(28)..

Bottles collected from

the M.S.C. creamery consisted of one of the first few
from each vat and it has been demonstrated that such
bottles have a higher incidence of coliform organisms.
Therefore, much importance should not be attached to the
incidence of coliform organisms in these positive samples.
Four samp3.es out of 96 gave all positive broth tubes in
the highest dilution and hence their counts could not be
compared with those obtained from solid media.
The coliform count ranged from 20 to 5,000 per 100 ml
of which about 75 per cent of the positive samples gave
a count below 5 0 0 .
Oomparison of coliform and standard plate counts
No definite correlation could be elicited from the
coliform and standard plate counts .
samples, in 20 ml portions,

Co3.iform negative

sometimes gave standard

plate counts higher than 100,000 per ml, while many times,
when the standard plate count was below 50,000 per ml,
thousands of coliform organisms were present..

This is

depicted in Graph II in which, to avoid plotting 96
samples on the graph, the arithmetic averages of the
coliform and standard plate counts of about every 5
samples, the counts of which were in a certain range
(e.g., coliform count from 100 to 1,000 and standard
plate count 10,000 to 1 0 0 ,0 0 0 ), were plotted.
It is therefore concluded that the standard plate
count of pasteurized milk Is of little significance as an

II

tandard plate Count

4

i

t
i
-i

A.
4

hi.
4

-L

oilform Count

TX.

17

-f •
'f. ■

Kumbar of Ga.rirole

21

26

index of improper pasteurization or recontaniinatlon.

It

has been found in the id.S.C. creamery that sometimes a
higher incidence of thermoauric and thermophilic bacteria
in raw millc results in a fairly high count in that milk
after pasteurization although the milk proved negative to
the coliform test..

This point contributes to the con­

clusion that there is no definite correlation between the
coliform and standard plate counts.

Such pasteurized

milk, on the basis of the standard plate count, may wrong­
ly be interpreted as rccontaninated or improperly pasteur­
ized..
Comparison of brilliant green lactose bile broth with two
solid media
The coliform counts of 90 positive samples were com­
pared with brilliant green lactose bile broth and desoxycholate agar.

Of these, 52 samples gave higher counts in

desoxycholate agar and 35 samples gave higher counts in
brilliant green lactose bile broth while 3 samples had
equal counts.

The average brilliant green lactose bile

broth coliform count and the desoxycholate agar coliform
count were determined.

Representing brilliant green lac­

tose bile broth as 100 per cent efficient,

the per cent

efficiency of desoxycholate agar (1 8 0 ) was obtained.
Seventy-four positive samples -were compared in
brilliant green lactose bile broth and violet red agar.
Twenty-five samples gave a higher coliform count in
brilliant green lactose bile broth and 46 samples in

27

violet red a.gar while 3 samples ga.ve equal counts.
Assuming brilliant green lactose bile broth to be 100
per cent efficient,
cent efficient

violet red agar proved 169.3 per

(see table 5 , page 2 8 )..

Comparison of brilliant green lactose bile broth and
formate ricinoleate broth
Fifty-one coliform positive samples were compared in
the two broths, namely brilliant green lactose bile broth
and formate ricinoleate broth.

Twenty samples ga.ve a

higher coliform count in brilliant green lactose bile
broth and 2 sarrroles in formate ricinoleate broth while 10
samples had equal counts.

These results were very closely

comparable but the per cent efficiency of formate ricin­
oleate broth
when

was 120.7 per cent (determined as above)

that of brilliant green lactose bile

broth was 1 0 0 .

It was noticed that formate ricinoleate broth produced a
more

copious amount of gas than brilliant

bile

broth in the same length of time*.

green lactose

As some members of the Salmonella group give false
positives

(3 3 ) in formate ricinoleate broth, all coliform

presumptive positive tubes of this medium, numbering 1 5 9 ,
were confirmed by transferring 4 loopfuls from each tube
into a brilliant green lactose bile broth tube and incu­
bating the latter at 37° C for 48 hours.

These brilliant:

green lactose bile broth tubes were all positive although
some of them formed only a bubble of gas.

From each of

the tubes having a bubble of gas a trace of culture was

28

streaked on eosin methylene blue agar plates and produced
typical coliform colonies showing thereby not a single
false positive in formate ricinoleate broth.
TABLE 5
Goinparison of Standard Liquid and Solid Media Used for the
Presumptive Coliform Test in Milk

Media

Per cent
Efficiency

Total Higher Equal

Average
Coliform
Count per
100 ml

20

172.1

100

No. of Samples

B.G.L.B.
broth

10

51
Formate
ricinoleate
broth

21

207.6

120.7

B.G-.L.B.
broth

25

162.3

100

74

3

Violet red
agar

46

274.8

169.3

B.G-.L.B.
broth

35

172.8

100

g

90
Desoxychol"
ate agar

52

312.2

180

Violet red
agar

24

274.8

100

326.7

119

io

74
Qesoxycholate agar

32

29

Comparison of violet red a;:-ar and desoxycholate agar
During the study of these two media it was experi­
enced that duplicate plates, each seeded with 1 ml of milk,
when compared to duplica.te plates, each seeded with 2.5 nil
of milk, gave a lower coliform count per ml; for this
reason 2.5 nil of milk per plate was used for solid media
thereafter.
Of 74 samples compared,

desoxycholate agar gave high­

er counts in 32 samples and violet red agar in 24 samples
while 18 samples gave equal counts..

When violet red, agar

was assumed to be 100 per cent efficient, desoxycholate
agar proved 119 per cent efficient (see table 5).,

These

results show that the two media are approximately equally
superior1 to brilliant green lactose bile broth or formate
ricinoleate broth, desoxycholate agar being slightly
superior to violet red agar..

In addition, desoxycholate

agar has the advantage of producing more and larger1 typical
dark red colonies.
Colonies picked by random selection from violet red
agar plates representing 67 samples were transferred to
brilliant green lactose bile broth and subjected to the
Completed Coliform Test.

It was found that of the 186

violet red agar colonies picked, 97 per cent were coli­
form cultures.

Under similar conditions, 383 desoxy­

cholate agar colonies, representative of 83 samples, after
the Completed Test,

gave 96.7 per cent coliform cultures..

These results showed how few false positives these tvro

30

solid media produced
One noticeable point was that colonies having less
than 0.5 mm diameter (which the author calls pin point
colonies) occurred very rarely in positive plates of both
media..

These pin point colonies will be described later.

A comparison of the detection of different Il-IViC
types in 48 coliform positive samples was made with desoxy­
cholate agar and violet red agar.

About 3 colonies each

from violet red agar and desoxycholate agar plates, of a
positive sample, were niched at random and purified cul­
tures were obtained from them.

One hundred and thirty-

one cultures from desoxycholate agar plates and 131 from
violet red agar plates

(representing 48 positive samples)

were typed by II-IViC reactions and 11 ILiViG types were identified.

A comparison of the numbers of cultures b e ­

longing to one type and recovered from each medium showed
that practically all the 11 types were equally detectable
on both media, excepting 2 "Intermediate" types, namely,
+-++ and ++++, which were not at all detected by desoxy­
cholate agar..

The number of times one type occurred in

48 samples in one medium was also found comparable with
that obtained on the other medium (see table 6 )..

Thus It

was concluded that desoxycholate agar and violet red agar,
in general, are about equally good for growing coliform
bacteria, when present in small numbers in pasteurized
milk.

31

TABLE 6
Comparative Detection of IMViC Types in 48 Coliform P o s i ­
tive Samples by Violet Red Agar and Desoxycholate Agar

II-IViC
Section

IMViC
i.y pe b

No.. of Samples
(out of 48)
Positive on

No. of Cultures
Obtained from

Des.
agar

V-Red
agar

Des „
agar

V-Red
agar

16

12

26

20

6

6

9

6

-+-+

25

25

41

38

+- ++

0

3

0

3

++ -+

1

2

1

3

+ + ++

0

1

0

1

-----

2

1

4

1

- + + ■*-

2

1

3

3

— ++

21

23

32

39

+

10

13

11

15

+~

2

2

4

2

89

131

131

---

E. coli

"Inter­
med­
iate"

A.aerogenes

---

—

Total---

--85

32

Superiority of desoxycholate agar over brilliant green
lactose bile broth
All these comparisons of desoxycholate agar and
violet red agar showed that there was little choice b e ­
tween the two media.

However,

desoxycholate agar, giving

a higher per cent efficiency, and la.rger, typical, dark
red colonies, was adopted as the best solid medium for the
Presumptive Coliform Test..
A further comparison of desoxycholate agar with
brilliant green lactose bile broth was thus made to d e ­
termine the superiority of the former*.

Of 90 positive

sanroles, the numbers of samples having coliform counts
below 1 0 , 3 0 , 5 0 , 1 0 0 , 2 5 0 , 5 0 0 , 1000 and 5000 were d e ­
termined in desoxycholate agar and brilliant green lac­
tose bile broth.

As desoxycholate agar gave a higher

coliform count than brilliant green lactose bile broth
in milk, it usually gave a smaller number of samples b e ­
low a certain coliform count than did brilliant green
lactose bile broth (see table 7 , -page 3 3 ).
Plotting the number of samples according to the
group in which they occur,

curves

for brilliant green

lactose bile broth and desoxycholate agar were obtained
as shown in Graph I I I .
A study of these curves showed that the greater bulk
of samples had coliform counts between 30 and 500 and it
vias in this range that desoxychola.te agar proved far
superior to brilliant green lactose bile broth.

Below

ml

00

De 8o ;-:yoho 1 a te Agar

100

Brilliant Green Broth
-z

10

60

a coliforn: count of 30 and above a coliform count of 5 0 0 ,
brilliant ^reen lactose bile broth and desoxycholate agar
tended to give parallel counts.
TABLE 7
The Numbers of Samples below Certain Coliform Counts as
Obtained by Two media

Col if or;:..
Pooltive
Samples

Colif orm
o o ant
Below

h 0 » of 0nmoles in
B,G.L.B . broth

Dssoxy,. agar

10

9

9

30

25

23

50

39

32

100

55

43

250

75

51

500

81

78

1,000

85

82

5,000

90

90

90

Percentage distribution of IllViC types and sections
Of 92 positive samples,
obtained.

445 coliform cultures were

All these purified cultures had been derived

from original,

typical, dark red colonies selected at ran

dom from violet red agar and desoxycholate agar plates

34

and sxitojacted to the Completed Coliform Test.
tures were typed by II-T71C reactions

The cul­

(59) which had been

generally utilized by workers in the past two decs.des.
The per cent distribution of a type was determined
by calculating the average of various percentages in which
that type occurred in different samples.

tleven types

and their respective percentages and the percentage of the
coliform sections, viz.. E . c o l i , "Intermediate" and
A. a e r o g e n e s . to which those types belonged (1) are given
in ta.ble 8 .
The "Intermediate" having the IhVIC formula -+-+ was
the most prevalent type, 30 per* cent of the cultures being
of this t y p e ,

In 1932, b'erkman and Gillen

(49) placed

this organism in the genus Citrobacter and 3erp;ey1s manual
of Determinative Bacteriology
Escherichia fr eun dll .

(56) classifies it as

On eosin methylene blue agar plates

this type gave colonies with a bright, metallic sheen and
large,

dark centers resembling very much the typical

E. coll colonies.

The highest percentage of the "Inter­

mediate" section of coliform organisms in pasteurized milk
was largely due to this coli-like type.
Four "Intermediate" types which showed no "inverse
correlation"

(53) of the Voges-Proskauer and methyl red

tests, being either positive or negative to both, con­
stituted 8.7 per cent of the total type percentages.

They a,re expressed as non-inversely correlated "Intermediate"
types In ta.ble 8.

35

TABLE 8
Percentage Distribution of Various Types and Sections of
Coliform Bacteria in Pasteurized Ailk

% NonPosi tiv© Gul Lares Coli foral I--.ViC Type Section inversely
cf
o cLiiro-l-o s Typed
Sections Type s %
correl­
7°
ated

OJ

++ —

445

•
o

92

E .. coli

27.-6
— +—

7.4

-+- +

30.8

+ —+ +

4.3

++- +

2.2

"Inter­
mediate "

41.7
1.5

1.5

1.5

1.5

-+ + 4-

1.4

1.4

—

25

+ + + +

A. aeroKenes

- —

—

Total----

+ +

+

+

4.3

_

1.4
-100

30.7

4.3

100

8.7

36

The majority of the samples contained two or more
Ii-iVIC types representing at least two sections,.

However,

in 27 per cent of the samples only single types were identlfied,.

IAViC type -■*--+ was present in 9.-8 per cent

of the samples,

++-- in 13 per cent of the samples and

--++ in 4,3 per cent of the samples.

Similarly,

In 37 per

cent of the samples only single coliform sections i.e.
E . c o l l , !iInternedi a t e " , and A. aero gene 3 were found in
13 per cent, 18,5 per cent and 5,4 per cent of the samples
respectively.

In every respect the most prevalent type,

as determined in 92 positive s ampl es, was the
ate " - + - +, next were types ++—

"Intermedi­

and — ++ (see table 9,

page 37}.
The number of samples

(out of 92} and the per cent

of samples from which a certain type was isolated are
shown on table 9 .
Comparison of the percentage distribution of coliform
sections determined by teo methods
hethod I
In this method,

generally used by other worhers,

only the cultures representing, definite coliform types
present in a certain sample were used for the determina­
tion of percentage distribution of coliform sections,
All the other cultures from the same sample, which on
identification proved duplicates, were discarded.
all,

In

208 such cultures were derived after identification

of 445 cultures from 92 positive samples,.

The percentage

37

distribution of various coliform sections was calculated
by the fallowing; formula..
'■{, distrlbution of

section

_

no., of cultures of section
total cultures (2 0 8 )
x

..nn

TABLE 9
Distribution of 11 ILV1C Types in 92 Coliforcu Positive
S arapl e s
I sol n'1rod. froui
ILVIC
Types

1:0.

Of

ypiivil (=*c..(

;J of
Sampl es

y of Samoles havinr 100 <
One
Type

One
Section

L. coli
+ + “-

~z.h.

37

9 .8

IQ

on «•v
<

C

60

13

13

"Inter-”
me di a t e "
- +-+
■+—+■+

OD
-

8.7

0

■i-1-- +

6

6.5

C

+ ++ +

5

5.4

0

4

4.3

0

h

4.3

0

48

52

4.3

19

20.7

c

-- j.„

6

6.5

0

Total

208

18.5

- + ++
A. aeroyenes
--+•+

27.1

5.4

36.9

38

Method II
In the second method employed in this work,
(an arbitrary number)

4- to 6

typical coliform colonies vfere

selected at random from 4 violet red agar and desoxy­
cholate agar plates representing 1 sample.
coliform cultures,

Purified

obtained from these colonies and con ­

firmed by the Completed Test, were typed by IMV1G tests
and the oercentage distribution of different coliform
sections in that sample was determined.

Ninety-two

samples ware examined as above and the average percent­
age for each section was determined.
The percentages determined by the two methods are
compared in table 10.

The second method gives a much more

accurate picture of the percentage distribution of c o n ­
form sections and is recommended.
TABLE 10
Comparison of Average percentage Distribution of Coliform
Sections by Two Methods
Average f0 Distribution Determined by
Sections

Method I
(208 cultures)

Method II
(445 cultures)

E.. coli

28.5

27.6

"Intermediate"

39.4

41 -7

A. aerogenes

35.1

30.7

Total

100

100

39

Pin point colonies
Pink colonies,

20 to 24 hours old and 0.5 mm or less

in diameter on violet red agar or desoxycholate agar, are
designated as pin point colonies.

These colonies did not

characteristically precipitate bile salts as is so common­
ly done by larger colonies..
Of thousands of colonies on 368 violet red agar and
desoxycholate agar plates representing 92 samples, only
39 were pin point colonies.
from 13 samoles,

Of these 39 colonies isolated

28 belonged to the mlcroaerogenic type of

aberrant coliform bacteria

(7 0 ) a s they formed a bubble of

gas within 3 to 9 days in lactose broth, while 11 colonies
did not produce any gas in 14 days.
No further study was made because of the insignifi­
cantly low incidence of these colonies on desoxycholate
or violet red agar plates seeded with pasteurized milk.
The source of coliform organisms in pasteurized
milk will be discussed under Vat Samples.
Vat Samples
An examination of 26 duplicate samples, drawn direct­
ly from vats in the R.3.C. creamery, was made in October,
1947.

Routine bottled samples of pasteurized milk, n u m ­

bering 23, were also collected in that month from the same
creamery and tested.

Five of these bottled samples corres­

ponded to 5 vat samples and will be discussed later.
The phosphatase field test was read on all the 26
samples, of which 21 produced upto 2 phosphatase units,

40

3 produced 2 to 5 units,
respectively.

and 2 produced 50 to 100 units

The last 2 samples were not properly

pasteurized as indicated by the phosphatase test and the
initial coliform count.

Two samples which gave 2 to 5

phosphatase units were positive in brilliant green lactose
bile broth before incubation of the milk.

Samples number

4, o, 1 1 , 13 and 20 (see table 1 1 ) were properly pasteur­
ized, as shown by 2 phosphatase units,

and gave no po si­

tives in brilliant green lactose bile broth before inc u­
bation.

They proved coliform positive after incubation

showing that at least one coliform cell per 100 ml did sur­
vive proper pasteurization.
TABLE 11
Phosphatase Test and Bacterial Counts Before and After
Incubation of Vat Samples

Sample
Number

Pho s phatase
Units

Coliform Count
per 100 ml

Standard Plate Count
per ml

Initial

Initial

Final

Final

4

2

0

340

2,100

180,000

6

2

0

300

22,300

5 ,000,000

7

in
i
OJ

51

10,000

29,000

2 ,300,000

8

2-5

22

9,800

85,000

1 5 ,200,000

11

2

0

1,090

32,000

4,200,000

13

2-5

0

1,800

4,600

600,000

18

100

180

u n s a t •.

441,000

unsat.

20

2

0

1,600

99,000*

242,000

21

50

180

unsat.

148,000

unsat.

*A great majority of colonies were pin point colonies.

41

All "the 26 samples were incubated for 6 to 8 hour's.
Initial and final standard plate counts were made before
and after incubation and a comparison of the counts showed
that the bacteria reproduced 6 to 8 times.

Initial and

final coliform counts also confirmed this.
Tyoes isolated and their percentage distribution
Samples 18 and 21 were not typed as no isolated col­
onies coyld be obtained from desoxycholate apar plates.
Eight colonies from a positive desoxycholate s.,par plate
of each of the remaining 7 sarnies were selected at ran ­
dom and purified cultures were typed by IMViC reactions.
None of the colonies proved to be a false positive.

Five

samples hard 100 per cent E. c o l i , with IiViC formula + +
and 2 samples had 100 per cent A. a ero penes, with IMVIC
formula

None of these samples contained more than

one type as shown in Table 12 (page 42).
Probable source'of coliform bacteria in pasteurized milk
Of 26 incubated vat samples 17 did not have a single
coliform cell in 100 ml while 1 out of 23 bottled samples,
from the same creamery,

examined during the same month

proved negative to the coliform tests in 20 ml.
Five vat samples did not have any coliform bacteria
per 100 ml but when 5 bottles,

corresponding to these vat

samples, were tested, 5 0 , 3 2 5 , 2 5 0 , 200 and 575 coliform
organisms per 100 ml were found and 2 to 4 IMViC types
were present In each sample.
In 92 ps,steurized samples only 27 p©r cent of the

42

samples had 100 ner cent of one type while 73 per cent of
the samples had two or more IkViC types.

None of the 7

incubate-d vat samples gave more than one type in each
sample showing thereby that if any cells survive after
pasteurization they belong to one type, predominantly of
Ii-iViC formula + +

None of the 7 incubated vat samples

had any "Intermediate" types but in pasteurized bottled
milk these were isolated from 60 per cent of the samples«
TABLE 12
Distribution of E.. coli and A. aerogenes Sections in 9
Incubated Coliform Positive Vat Samples

Total
Samples
Run

No,, of
Positive
Samples

26

Percentage Distribution
Cultures
Typed

E.- coli
(++--)

"Inter­
mediate"

A. aero­
genes (— M-)

4

8

100

-

-

6

8

-

-

100

7

8

100

-

-

8

8

100

-

-

11

8

100

-

-

13

8

-

-

100

18

u n s a t i s f

20

8

21

u n s a t i s f

100

a c t o r
a c t o r

y *
y .

It was therefore concluded that the presence of c o n ­
form organisms in pasteurized milk resulted from recon­
tamination .

43

The greater the number of coliform types present In
a bottled sample the more varied are the sources of contam­
ination .
DISCUSSION
The environment of the cow and the conditions under
which milk is produced and handled are such that the
presence of coliform organisms in fresh rs.w milk is al­
most universal.

Not one sample of 104 proved negative to

the coliform test when about 10 ml of milk was examined..
However, Slack and Kaddeford (72) reported that 14 per
cent of their samples of raw milk were negative to the
coliform test when 1 ml \?e.s tested and Bartram and Black
(7 3 ) found that 36.4 per cent of the samples which had
standard plate counts of 10,000 or less were free of c o n ­
form bacteria In 10 ml tested.

In the present study none

of the 29 samples having a standard plate count of 10,000
or less gave a negative coliform test, even though these
sarnies were properly refrigerated and tested within 3
hours after milking.

Either very sanitary production of

the milk or less sensitive media used seem to be the cause
of so many coliform negative samples in the hands of those
workers.
The author's observations are similar to Finkelstein1s
(74) who found that milk Initially contaminated on the
farm had 100 coliform bacteria per ml under careful pro­
duction and 5S8 per ml when no care was used.

Sherman

and ding (9) suggested that a standard of 100 coliform

44

bacteria per ml in high grade milk and 10 in certified
milk was not stringent.
In properly pasteurized milk a positive coliform
test as a result of the survival of heat resistant coli­
form organisms is highly improbable.

Of the 24 vat

samples tested only 2 samples were positive, having 51
and 22 coliform bacteria per 100 ml.

decontamination or

improper pasteurization were definitely the cause of the
presence of coliform organisms in bottled milk.
ChiIson

and

ooworkers

(24) stated that the coliform

test "should supplement and not supplant the standard
plate count" in detecting recontamination; but,

the present

studies revealed that of the 92 coliform positive samples
63 had standard plate counts below 30,000 per ml with a
logarithmic average of 9,000 per ml while of the 41 c o n ­
form negative samples 20 had standard plate counts above
30,000 per ml with a logarithmic average of 63,000 per ml.
Those 63 samples, which on the basis of the coliform index
were regarded as recontaminated,

might not be reported so

by the standard plate count while the 20 coliform negative
samples might rightly or wrongly be reported recontamin­
ated as a result of the standard plate count.

The higher

standard plate count might possibly be the result of
slight growth after pasteurization or the presence of
thermoduric and thermophilic bacteria in raw milk.
fore, the coliform index is more reliable

(18)

There­

(3 0 ) (1 9 )

(2 3 ) (2 0 ) in detecting recontamination than the standard

45

plate count which might be used to supplement and not
supolant the coliform test.
The media developed for the determination of coliform
organisms in water have, in the past, been used for the
coliform test in milk.

However, milk is rich in fat and

proteinacious matter* and behaves differently from water.
This v/as demonstrated by Barkworth (75) who inoculated
coliform organisms into milk and water at the same rate
but found tliat milk gave a lower percentage of positives
than w a f e r =

Raw milk possesses a germicidal prooerty

which may supplement the inhibitory power of a medium
when seeded with milk.
I-lcAuliffe and Farrell (3 8 ) recommended that the dye
concentration of brilliant green lactose bile broth be
increased 2.5 times of the standard concentration as it
is partly adsorbed by the solids in 1 ml of milk,.

This,

they claimed, would eliminate false positives.
Stark and Curtis

(71)

(35) introduced formate ric-

inoleste broth and claimed its superiority over brilliant
green lactose bile broth as it gave very few false positives in milk,

Farrell (37) reported that formate ricin-

oleate broth gave 100 per cent higher "l-dTT'1 than brilliant
green lactose bile broth in raw milk.

In the present,

study It proved slightly superior to brilliant green lac­
tose bile broth in pasteurized milk and did not give a
single false positive, but when compared with violet red
agar in raw milk it was found quite inferior.

46

The need for a good solid medium (7)
been felt*

Yale

(31) has always

(32) compared 10 solid media developed

for the enumeration of coliform bacteria and found d es­
oxycholate agar and violet rod agar to be the most p r o m ­
ising when 1 ml of milk was used and that duplicate agar
plates gave as reliable counts as 15 broth tubes.

The

superiority of desoxycholate agar has also been emphasized
by other workers

(39)

(41)

(4C)

However, Bartram and Black

(7 6 ).
(36) compared 2 strains

each of E s c h e r i c h i a , ’'Intermediate" and Aerobactor in
different solid media and graded desoxycholate agar as
sixth in the descending effectiveness of solid media,
ileutral red agar and violet red agar were placed first
and second..

They stated that the Inferiority of desoxy­

cholate agar m s
"Intermediate"

due to its inhibitory action against the

strains used.

In this study desoxycholate

agar proved just as good as brilliant green lactose bile
broth for raw milk but for pasteurized milk it showed a
marked superiority over the broths with a. slight margin
of efficiency over violet red agar.

No Inhibitory effect

of desoxycholate agar as compared with violet red agar
was discerned in the detection of the "Intermediate"
and other types of coliform organisms in pasteurized milk.
In pasteurized milk desoxycholate agar has the fol­
lowing advantages:
1.

It gives a higher coliform count.

2.

Up to 2.5 ml of milk can be added to each plate.

(Buchbinder has used 4 ml per plate of 15 ml a par-)
p.

It gives larger and more distinct typical, dark red
colonies than does violet red agar.

4.

Typical,

dark red colonies prove to be coliform

organisms In 97 per cent of the cases.
5.

The occurrence of atypical colonies

(less than 0,5

dm

in diameter) is rare and when they do occur the major­
ity of them belong to the aberrant coliform group*
The presence of this group indicates recontamination.
6.

It is most efficient when coliform organisms are
present in small numbers and so Is especially appli­
cable to pasteurized milk.

7«-

The results are available after 18 hours incubation*

8..

Duplicate desoxycholate agar pishes, each seeded with
2.5 ml of milk, gave higher counts than were obtained
with 15 brilliant green lactose bile broth tubes.

9*

It is as productive as violet red agar in detecting
all coliform types when present in small numbers in
milk.
On the basis of the above advantages it is strongly

recommended that duplicate desoxycholate agar plates,
each seeded with 2.5 ml of milk, be used for routine exam­
ination of pasteurized milk In Public Health Laboratories.
Tiedernan (39) and Buchbinder

(40) have recommended one

plate seeded with 4 to 5 ml of milk but it Is always safer
to seed duplicate plates than a single plate.
P e r r y ’s E. G. medium (77) to detect coliform bacteria

48

at 37° G and E. coll at 45.5° C and Leifson's

(78) newly

developed hyodesoxycholate agar and broth have been reported to give good results in the examination of milk.
A comparison of desoxycholate agar with these media for
testing pasteurized milk would be worth while.
In the determination of the percentage distribution
of coliform types three provisions should be met.
First,
place,

samples of mllk,

in which no growth has taken

should be employed so that the relative proportion

of various coliform types remains similar and truly repre­
sentative of the initial coliform contamination,

Malcolm

(79) seeded raw milk with bovine feces, containing predominantly E. c o l l , and after 36 hours incuba.tlon at
62.5° F found that the majority of coliform bacteria b e ­
longed to the A., aerogenes section.

This shows that high*

er storage temperatures may change the proportion of coli­
form types from that initially present;

therefore,

the

source of contamination may be interpreted inaccurately.
Second,

pour-piste cultures should always be used

for typing because in broth cultures one type may over­
grow the other types.
Third, random selection of colonies from a pourp l a t e , rather than Isolating the different types from a
sample,

should be used.

Many authors

(30)

(35)

(37)

(69)

(76)

(80)

(81)

have determined the percentage distribution of coliform
sections but as they did not follow the above three

49

provisions their percentages cannot be compared with the
percentages found in this study*.

In aditfion,

the dis­

tribution of coliform bacteria in milk differs according
to l o c a l i t y , climate and conditions of production.
The percentage distribution of coliform sections
has rarely been studied in pasteurized milk..
Levine

Vaughn and

(56) suran.:.-I zed the incidence of "Interraediate11

becterla in focos, u r i n e , soil,

milk, eggs, etc.,

and in

none of thorn wss the percentage distribution more than
9 9 .5 ,.

In the nresent study 41 per cent of the coliform

bacteria in pasteurized milk belonged
section.

to the "Intermediate”

In lg per cent of the samples the "Intermediate"

tyre - + -+• ua s the only coliform. by p ?• -'-resent.

The cause

of this eery high incidence of the "intem o dio.te" section
could not. be -ascertained.

hinha w itsch and cowoi"-kers {0 ~ )

repo-f sd the change of L « coil , seeded in s o i l , into
citr-'te utilizing "Intermediates” .

It -would be interest­

ing to do ter nine if under dairy plant sanitizing -procedures
some h. coll change into

"Intermediates” «

Of the 16 possible I,:ViC typos, 11 from pasteurized
and 13 from raw ;..ilk v.nro Isolated.

Throe types

with

IkViC formulae * + +-,

+- + -and 4---- v/ere not Isolated in

this study.

(8 3 ) reported

Sanborn

the isolation of all

the 16 typos in a study of slime producing coliform
organisms.

Ruchhoft

and coworkers

4 fecal coliform types,

++--,

(,84) asserted that only

- +— , -+-+ and — +4, exist

and the others are either mixed cultures or extraneous

to feces,.

Wilson and coworkers

(7) stated that cultures

which show 110 "inverse correlation" of Voges-Proskauer
and methyl red reactions are mixtures,.

Later workers

(54)

(60) have consistantly found non-inversely correlated
cultures,

some of which, after numerous serial plantings

in lactose broth for rjurification, have shifted to in­
versely correlated cultures while the majority of the is
have proved non-invorsely
era

correlated,

Stuart and cowork-

(54) reported about 10 per cent of non-inversely

correlated cultures in 1353 raw samples that they examined
and in the present work 3,7 per cent of these types in
pasteuriz,ed and 11.75 per cent in raw milk have been found,
Parr classifies them in the "Interiaedlate" section but
ilitchell and Levine

(57), on the basis of the dissimila­

tion of nucleic acid degradation products, have grouped
them with the A.. aero .genes section.
with Parr's classification.

The author agrees

Stuart and coworkers

(54)

described the inversely correlated types ++++■ and - + + + as
"Irregulars".
This study shows that the presence of aberrant co li­
form bacteria in pasteurized milk is rather infrequent
but in raw milk they constituted 13 per cent of the total
coliform count and the IkViC type -++- was the most p r e ­
valent.

It is interesting to note that this anaerogenic

type was never isolated from pasteurized milk.
The Standard Methods for the Examination of Dairy
Products is not designed to detect these bacteria and

51

their insignificant presence in pasteurized milk makes
them of no importance to dairy bacteriologists.

Besides,

their sanitary significance has yet to be determined.
ir
Ostman (8 5 ) found that 47 of the 50 Friedlander1s
bacilli cultures, belonging to serological types A, IB,
G and X, gave biochemical reactions, such as acid and gas
production in glucose end lactose .and IkViC reactions,
oharac teri sti c of typical and aberrant coliform organi sms .
He concluded that "valid criteria have not yet boon estpM

1 shed Cor the differentiation of orr.anisms of the
tt

Friedlander and coli-aerogen.es group

|t

.

In the aosence
u
of bioch emi c•:1 tests to differentiate Fr ied lan der 1s

bacilli from the Escherichia-Aerobacter group our understanding of the pathogenicity of coliform bacteria is not
c o mpl ete .
Standard methods for the Examination of Dairy Products
(11) regards the presence of all coliform organisms,
both fecal and non-fecal origin,
unsanitary dairy practices.

of

as direct evidence of

It is immaterial whether the

presence of E. coll is the result of direct or indirect
(as through utensils)

fecal contamination of milk.

The

occurrence of the S. coll section in pasteurized milk is
decidedly not directly fecal but due to growth of these
bacteria In nooks and corners of equipment that escape
usual cleaning practices.

The A. aerogenes section is

non-fecal but the "Intermediate" section is found In
small numbers in human and animal, feces.

Parr

(,60)

no

report,eel 15.3 per cent of this section in human feces.
FroqTiont occurrence of two or mox*e typos in pasteurized milk Indies tes varied sources of re con tumi na ti on 0.0
from (0 6 ) v a t s , valves, p u m p s , pipes, cooler, hoir-ogeni zex*,
bottler, bottle and the personnel and reflects Improper
cleaning ox-ac tices.

Bottled s a roles containing two or

more types generally had a hi gher 3 nciOunce of col iform
bacteria.

Presence of only ore type shows a possible

bacterial pocket of contanination somewhere in the equip;iert.

The rax-o occurrence of only one coliform type in a

raw sarnie chows a diversity of sources of c on taivdnc ti o n .
I Oh
Raw hi Ik
1.

A hi ah coliform count vns concurrent with a high
standard plate count and vice versa.

2.

Sonnies from the same producers when examined in
summer gave s. higher coliform and standard plate
count than in ’winter.

p.

Desoxycholate agar proved equal to brilliant green
lactose bile broth with 53 samples while violet red
agar gave a 1.6 times higher average coliform count
than formate ri cinolec.be broth with 30 samples.

4.

F rom 55 samp]es, 553 cultures were selected at random
and utilized for the determination of the average per
cent distribution of coliform types and sections.
44.e E. coli section constituted 23*5 par cent,

"Int­

ermediate" 21..9 per cent, and A. aerogenes 54.8 per

Of

cent.
About 12 per cent of the c aliform organisms present
in m.ilk belonged to non-inversely correlated IilViC
types.

Of Farr's 16 possible Il-.ViC types 13 were

isolated from 55 samples and the Il-IViC type --++
constituted about 48 per cent of the total percentage
of coliform types.
Aberrant coliform bacteria composed 13 per cent of the
total colifor:. count,
IJ \J

X

A,

the most orevrlent.

x i

4.5 per cent being rlcroaerov.ri th I i_Yi C tyrje - +-

V

None of these aberrant coliform

organ! s:..s belonged to too A. coli

section.

No definite source of con lamination could be ascer­
tained .

However, in 6 of the 36 samples the A. coll

tyne + + -- composed 92 to ICC -per cent of the coliform
count and indicated direct fecal contamination.
Paster rl zed I-Illk
No def ini t

corr o 1 a ti o n could bo as tabli shed be tween

the standard plate and co1 ifora counts In 96 coliform
ocsit.ive ca m u l e s , showing thud the standard plate
count as an index of r 1contamination is not depondable but the cot.if01 a in Iox is reliable and the former
'b suor.! ant the let ter

any sun 0! enon t. b:.
v

—

udy of media v; .m

the aer cent efficiency of

brilliant gr een lactose bile broth,
a
11

one

covrroa m o

1 on
nor

>

e m u

.. n* re -•

*

arbitrarily chosen

to ether media it was found that
,-

11 i 1. c - ; u r ,

...

am

formate ricinol mate broth

54

was 121, violet red ayor 169, and desoxycholate ar.ar

10. As desoxyeholato ayar a.aue 1.5 fines hiyher a v o r y e
coliform count than brilliant yreen lactose bile
b r o t h , it is tentatively r-ocomrended that duplicate
desoxycholate soar pl a t e s , seeded with 2.5 r.il of ailk
each, be used for the routine e v a l u a t i o n of bottled
milk in Public Health Laboratories.
“
1~ Average percentaye distribution of coliform typos and
A. -1- »
sections was determined in 92 coliform positive
samples.

The coli-like "Intermediate" type - + --t was

the most prevalent

(31 per cent) of all the 11 IliViC

tV p3 3 is o1 a,t q d .
1 2 . The non- inversely c o r rel ated IhViC types c onstituted
8.7 oer cert of the total perconto ye of coli for m

13

* r.o tyres
were
o-rr
\v
a.

oresent in 27 per cent of coliform

positive samples while two or mere types were present
in 73 per cent.
A few pin point colonies

(less than 0.5 mrn in diameter)

vrere observed on desoxycholate ay.?r and violet red agar
plates and a majority of them belonged to microaeroyenic aberrant coliform bacteria.
All vat samples which proved coliform positive only
after Incubation,

contained only one II-IVAC type per

sample but 73 per cent of the bottled samples showed
two or more types which indicated reconlamination.

crc

16. It w .b concluded that the greater the number of coliform; types preeent in a sample the m o r e varied are the
sources of contamination.

56

LITERATURE CITED
1.

Parr, L. W.3, 1-48.

1939

Coliform bacteria.

Bact... Rev..,

2.

Breed, R. S., and Norton, J. F.. 1937 Nomenclature
for the colon group.. Am.. J.. Pub. Health, 27, 560-5 6 3 •

3.

Standard Methods for the Examination of Dairy Products.
1941 Eighth Edition.. Am.. Pub.. Health Assoc., New York.

4.

R a w l a n d s , A.
1939
The udder as a possible source of
coliform organisms.. J . H y g . , 39, 456-462.

5.

Murphy, J.- 24., and Hansen, J. J.
1943
Infection of the
bovine udder with coliform bacteria*. Cornell Vet., 33,
61-77.

6.

Kon, P.. 24.
feces.
J.

7.

Wilson, Q-. S., Twigg, R. S., 7/right, R.. C., Hendry,
C. B., Cowell, M. P., and Maler, I.
1935
The bacteri­
ological grading of milk.
Med. Research Council, Spec.
Rept.. Ser. No. 206, London.

8..

Milk Ordinance and Code, Public Health Bulletin No.
220.. 1939
U. S. Pub.. Health Service, Washington, D.C.

9*

Sherman, J. M., and Wing, H. U.
1933
The significance
of colon bacteria in milk, with special reference to
standards.
J. Dairy Sci., 16, 165-173 ••

1933
Coliform organisms in milk
Dairy Research, 4, 206-212.

and bovine

10*. Yale, M. W., and Eglinton, R*. 1935 EscherichiaAerobacter group.. Ann. Rept.. Intern. Assoc. Dairy
and Milk Inspectors, 24, 116.
Reviewed by Tanner,
F. 7/.,Microbiology of Foods, Second Edition. 370. 1944.
11. Standard Methods for the Examination of Dairy Products,
In press.
Ninth Edition.
Am. Pub. Health Assoc., New
York.
12. Ayers, S. H., and Johnson, W • T.
1915
Ability of
colon bacilli to survive pasteurization.
J. Agr.
Research, 3, 401-410.
13.. Johnson.
1926 Second Ann. Rept.. Penn.. Assoc.. Dairy
and Milk Inspectors, 122.
Reviewed by Tanner, F. W.,
Microbiology of Foods, Second Edition,
129-130.. 1944.

57

14. Tanner, F.W.., and Windsor, M.. W.
1929 The ability
of Escherichia coli and Serratia marcescens to survive
63*8° C for thirty minutes in milk.. J. Dairy Sci..,
1 2 , 202 -2 1 0 .
15* Beavens, E. A.- 1930 The effect of lactose on the
survival of Escherichia coll when heated to 145° F
for thirty minutes.. J. Dairy Sci.., 13, 91-93 .
16. Long, H. F.., and Hammer B. W.
1944 Heat resistant
coliform organisms with particular reference to
butter.. J.. Milk Tech.., 7, 20-25.
17. Shippen, L. P.
1915
The significance of B. coli in
pasteurized milk.
J.. Am.. Med.. Assoc.., 64, 1289-1291.
18. Swenarten, J V C . . 1927
Gan B. coli be u s e d as an index
of the proper pasteurization of milk?
J.. Bact.., 13,
419-429.
19«.McCrady, M.. H.-, and Langevin, E.-H.
1932 The coliaerogenes determination in pasteurization control.
J. Dairy Sci.., 15, 321-329.
20. Barkworth, H.
1942
Coliform organisms and pasteur­
ization.. The Army Vet., Bull.., 36, 311-325*
21. Breuckner, H.-J.
1939
Significance of Coli in milk..
Am. Prod. Rev., 88: 27, 838 -8 3 9 . J. Dairy Sci., 23,
A80-A81.
1940.
22. Craig, J. E.
1946
Significance of concentration of
coliform organisms in raw milk upon survival of
pasteurization.. J. Milk Tech.., 9, 191-196.
23. Allexander, W. J., and Milone, N. A.- 1936
The sig­
nificance of the Escherichla-Aerobacter group in
pasteurized milk.. J. Bact., 31, 574.
24. Chi Is on, W . H . , Yale, M*. W.., and Eglinton, R.
1936
Detecting recontamination of pasteurized milk by
bacteriological methods.
J V Dairy Sci., 19, 357-343*.
25. Stark, C- N., and Patterson, M. C.
1936
The heat
resistance of colon organisms in milk.
J. Dairy Sci.,
19, 495-496.
26. Morris, C. S.- 1945
The presence in raw cow's milk of
a bactericidal substance specific for certain strains
of coliform organisms and comparative rate of growth
of bacteria in raw and pasteurized milk.
Dairy Industry,
10, 180-181.
Mature, 1 5 5 , 22. 1945.

58

27- Robinton, E. D . , and G-enung, E. F - 1945
The effect
of temperature on coliform organisms in milk and cream.
J. Milk Tech.., 8, 97-100.
28. Dahlberg, A. C.
1946
The relationship of the growth
of all bacteria and coliform bacteria in pasteurized
milk held at refrigeration temperatures.
J. Dairy
Sci., 29, 651-656.
29. Kessler, M . A . , and Swenarten, J. C.
1927
G-entian
biolet lactose peptone bile for the detection of
B. coli in milk.
J.. Bact.., 14, 47-53.
30. Kline, E. K.- 1930
The colon group of bacteria in
milk.
Nineteenth Ann.. Rept.. Intern.. Assoc. Dairy
and Milk Inspectors, 6 8 -8 5 .
31* McOrady, M. H., and Archambault, J.. 1934 Examining
dairy products for members of the EscherichiaAerobacter group.. Am. J. Pub. Health, 24, 122-128.
32. Yale, M. W.
1937
Comparison of solid with liquid
media as a means of determining the presence of
lactose fermenting bacteria in pasteurized milk..
Am. J. Pub. Health, 27, 564-569.
33. Stark, C. N., and Curtis, L. R.. 1936 Evaluation of
certain media for the detection^colon organisms in
milk.
Am. J. Pub. Health, 26, 354-356.
34.

Bartram, M. T., and Black. L. A.
1936
Detection and
significance of the coliform group in milk*. I. A
comparison of media for use in isolation*. Foo d R e ­
search, 1, 551”562.

35. Bartram, M. T., and Black.-L. A.. 1937
Detection and
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1939 Productivity
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37.. Farrell, M. A. 1937
A comparison of ten presumptive
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'1938 Dye concen­
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59

39- Tledeman, W. D., and Smith, S. E.
1945
Practical
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The keeping quality of p a s t e u r ­
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50. Malcolm, J. F.
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1938 Hi trogen a v a i l ­
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Sanitary significance of the
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61

63- Mallmann, W . L., and Darby, G. W.. 1941 Uses of a
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1938
A rapid phosphomonostearase test
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The Vog es-Proskauer and methyl red reactions in coliaerogenes grouo.
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1943
Notes on the use of s o d i u m - l a u ^ l - s u l p h a t e tryotose br o t h for the detection of E. coli in milk.
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69. Griffin, A. M., and Stuart, G. A.
1940 An ecological
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1940
Suggested grouping of slow lactose fermenting coliform
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71. Stark, C. N., and England, C. W.
1935 Formate ricinoleate b rot h -— a n e w medium for the detection of colon
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72. Slack, A. J., and Maddeford, G. W.
1932
The B. coll
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73. Bartram, M. T., and Black, L. A.- 1937
The detection
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J. Dairy Sci., 20, 105-112.
74. F i n k e l s t e i n , R.
1919
Occurrence of Colon-Aerobacter
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J. Dairy Sci., 2, 460-482.

62

75« Barkworth, H.
1943
Comparative detection of coliform
organisms in milk and water.
J. Hyg., 63, 129-135
76. Stuart, C. A., Wheeler, K. M . , and Griffin, A. M.
1938
Coliform organisms in certified milk.
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77. Perry, C. A., and Hajna, A. A.- 1944 Further evaluation
of E..C. medium for the isolation of coliform bacteria
s-11*3- E. ■c o l i .. Am.. J .. Pub. Health, 34, 735-738.
78. Eeifson, E.
1943
Hyodesoxycholic acid media for
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79. Malcolm, J.. F.
1939
The enrichment of Aerogenescloacae types in milk he ld at low temperatures; with
observations on the relative rate of growth of
Aerogenes-cloacae and B. coli types in milk at d if­
ferent temperatures.. J. Dairy Research, 10, 410-425.
80. Oeser, H.
1937
Bacterium Coli in der M i c h . . Zentr.
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81. Yale, M. W.
1933
The Escherichia-Aerobacter^group of
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82. M n k e w i t s c h , I. E., Rabin^witsch, D. J., and Joffe, F. S.
1936 tlBeitrage zur Frage uber die Herkunft un d die
sanitare Bedeutung der zitratassimilierenden Abarten
von Bacterium c o l i . Zentr. Bakt. Parasitenk., I.,
Orig^, 137, 152-160.. Reviewed in Bact. Rev.., 3, 1-40.
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8 3 . Sanborn, J. R.. 1944 Slime producing coliform and
coliform-like bacteria.. J. Bact., 48, 211-217.*
84. Ruchhoft, C. C., Kallas, J. G., Chinn, B., and
Coulter, E. W.. 1931
Coli-aerogenes differentiation
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8 5 . Ostman, E.
1949 A comparative study of coli-aerogenes
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Coliform
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