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TYPES OF GROWTH RATES
EXHIBITED BY HEAT RESISTANT ORGANISMS

ISOLATED FROM MILK

by
FRANK ROBERT EEABODY

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Bacteriology and Public Health

1948

THESIS

ACKNOWLEDGMENT

The helpful suggestions and kind assistance
of

Dr. W. L. Mallmann
faculty member
of the
Department of Bacteriology and Public Health
of
Michigan State College

are greatly appreciated

199540

“—9

OUT LINE

I. Introduction

A. Current use of thermoduric counts for determining

farmsanitation............................

B.Definitionofterms.......................

C. The problems encountered in interpretation of ther-

moduric counts in relation to total counts . . . . . .....

D. Purpose of the paper - to study the fundamental
behavior of thermoduric organisms by means of

growthcurves..........................

II. Literature Review

A. Isolation of organisms from pasteurized milk ..... . .

B. Use of laboratory pasteurization
1.Asacontrolmeasure ....... ..

2. Revealed discrepancies between thermoduric

countsandtotalcounts.................

III. Experimental Procedure

A. Selection of organisms for study. . . . . . . . . . . . .

B. Methods of laboratory pasteurization. . . . . . . . . . .

C.Growthcurves. .....

1. Preparation of inoculum

2. Techm'c of testing

3. Standards and temperatures used
4. Media used

5. Cultural and biochemical characteristics of the
cultures studied

10

IV. Results
A. Use of the “growth coefficient” . ................
B. Data on growth rates. ......... . ........... . . .
1. Growth curves at 37° C.
2. Growth curves at 250 C.
3. Growth curves at 510 - 55° C.
4. Growth curves at 610 - 650 C.
5. Growth curves at 50 C.
C. Correlation of the growth curves ........ . ........
V. Discussion

A. Division into three groups on the basis of growth
StUdiGS. 000.00.000.00 0.000.000.0000...

B. Possible explanation of discrepancies in thermoduric
counts from the results of the study. . . . . . . . . . . . . . .

C. Importance of growth rate studies and need for
furtherwork... ...........

VI. Conclusions . . . . ............. . . . . . ...........

VII. Literature Cited ....... . ............... . ......

ll

12

16

17

18

19
20

21

INTRODUCTION

The introduction of tryptone glucose extract agar as a standard
plating medium in the Standard Methods for the Examination of Dairy
Products (1) increased the total counts to a marked extent. This
increase in count was due in part to the appearance of the thermoduric
bacteria which were present only to a limited extent on the former
standard agar. The result was a marked increase in counts of pas-
teurized milk from sources high in thermoduric organisms.

In order to maintain bacterial counts of pasteurized milk within
the limits set by standard ordinances, it was necessary for the dairy to
eliminate the thermoduric bacteria from the milk. Although the organ-
isms are found on the dairy equipment, the chief source is producer
milk. Accordingly, considerable research was conducted on this group
of bacteria and standards were set by various research workers on the
maximum numbers allowable.

Thus, it is current practice for milk sanitarians and others
interested in dairy sanitation to make routine examinations for thermo-
duric organisms in milk as an aid in determining the causes of poor
quality. The thermoduric count of milk is used as an indicator of the
sanitary conditions of the equipment while high raw milk counts are
supposedly due to faulty cooling. When a producer’s milk shows a high

incidence of thermoduric organisms, it is customary to make a field

inspection. Generally, the field man finds dirty equipment. PrOper
cleaning, coupled with effective cooling, produces a good quality milk.
Such field observations have established the belief that the presence of
thermoduric organisms are evidence of dirty equipment. Thus, it could
be assumed that a low thermoduric bacteria count indicates good sani-
tation. The field man or sanitarian evinces little interest in the pro-
ducers of low count milk.

One of the first things to be considered in any discussion of
thermal effects on bacterial growth is a definition of terms. The term
“thermoduric mesophile” was coined specifically to describe those
organisms in milk which were found to be heat resistant. The expres-
sion does not necessarily indicate a time and temperature relationship
or the ability of the organisms to reproduce at pasteurization temper—
atures. There appears to be no exact definition of the term “thermo-
duric” even though it is widely used in the literature.

In this thesis the term “thermoduric” will be used to designate
all organisms surviving pasteurization irrespective of whether they
merely survive or actually reproduce at a pasteurization temperature
of 14.30 F. Gaughran (5) classifies thermophilic bacteria as those
organisms which have an optimum temperature of 50° - 60° C. How-
ever, for convenience in presentation, no distinction will be made
between thermoduric and thermOphilic organisms except as indicated

by actual growth curve data.

About three years ago, a field study of fifty producers was
initiated by the Department of Bacteriology, the Lansing Department of
Health and a local dairy to evaluate a number of different methods of
sanitizing milking machines. The study was reported by Mallmann, et
al, in 1946. (l l). The program extended over a period of 12 months.
Data were collected on the total counts of the raw milk and the thermo-
duric counts after laboratory pasteurization. These extensive data from
which the above report was taken, offered an opportunity to study the
correlation between sanitary practices and milk counts--both raw and
thermoduric--under actual field conditions. The information was
especially valuable since it was an unselected group of producers whose
sanitary practices were being observed but not controlled.

One of the most striking features of this report is the irregu-
larity with which high counts of thermoduric organisms are encountered.
This is true of both the good and poor producers. For example,
sporadically, a good quality milk with a low thermoduric count may
show for a day an unusually high incidence of thermoduric organisms
followed by a return to the low count. A high total count milk may have
a low thermoduric count which still shows the same irregular increases
in thermoduric indices as a good quality product. This is demonstrated
by the data from two selected milk supplies representing a good and a
bad producer. The total and thermoduric counts for several weeks are

presented in Table I. (10).

Table I. A comparison of the records of a good producer and a poor

producer expressed as organisms per ml.

Date

1/2
1/10
1/24
1/31
2/7
2/21
3/7
3/20
4/3
4/17

Logarithmic
Average

Producer “A”

Total
Count

10,000
20,000
12,000
31,000
19,000
21,000
32,000
10,000
52,000

19,950

Thermodur ic

Count
3,300
5,000

11,000
4,200
6,000
2,900
1,100
1,200

500

2,740

Producer “B”

Total
Count

38,000
100,000
250,000

1,700,000
140,000

164,000

1,560,000
12,600,000

476,300

Thermoduric

Count

600
2,900
800
800

27,000

1,300

1,800

1,100

1,740

Producer “A” had a consistently good quality raw milk with a
logarithmic average count for this period of 19,950 organisms per ml.
The record of Producer “B” was much lower in quality with the
logarithmic average of 476,300 organisms per ml. for this period.
However, in the case of the thermoduric counts the situation was quite
different. In the record of Producer “A” the highest thermoduric
count--almost twice as large as the second highest--is found in the
same sample as one of the lowest total counts. And it also happens that
the highest total count came from the same sample as the lowest ther-
moduric count. Examination of the data on Producer “B” gives even
greater discrepancies. The wide variations in total counts are not
reflected in the thermoduric counts. While the highest number of
thermoduric organisms is found in one of the better samples, the three
lowest thermoduric counts (which are essentially the same) came from
samples having total counts of 38,000, 250,000 and 1,700,000 organisms
per ml. It may also be noted that the average total count of Producer
“B” is about 25 times that of “A”, but the thermoduric counts are of
the same order. In fact, Producer “A” shows the higher average
thermoduric count.

Although these two are selected cases they are representative
of other producers studied. It is not to be implied that all of the data
are as striking as the instances cited, but these variations are of
sufiicient frequency to warrant serious consideration. Statistical

analysis of these data now in progress may show similar trends between

the two but from a practical standpoint, these marked fluctuations and
lack of correlation must be considered and explanations be found. In
each of four groups of producers studied, the three best and three
poorest were selected. The counts of the twelve producers in each of
these classifications were averaged and the following results obtained:
Organisms per ml.

Total Thermoduric

count count
Three poorest in each group 290,000 2800
Three best in each group 48,000 1100

These data show that the thermoduric counts are not essentially
different for the two groups. This substantiates the observations made
on the averages of the two producers with these data on 24 producers
for a period of 12 months.

This all added up to the fact that there were many observations
of the behavior of thermoduric organisms which could not be explained
on the basis of known facts. Why does a supply of milk which has low
counts suddenly show a high thermoduric count without a corresponding
increase in the total count? Should the thermoduric counts parallel the
total counts, should they remain fairly constant for all samples or are
the two entirely independent? What explanation can be offered for one
supply which has a high total count with a very low percentage of ther-
moduric organisms while another may be composed almost entirely of

thermoduric bacteria?

The routine determination of thermoduric organisms in milk
seemed to offer little help in solving these questions. It appeared that
some fundamental studies on the thermoduric organisms, particularly
as they pertained to growth patterns at various environmental temper-
atures, would be valuable. This thesis presents the results of a study
planned to furnish some of this basic information on the thermoduric

group of bacteria.

LITERATURE REVIEW

Many organisms have been isolated from pasteurized milk by
various workers. These organisms are cited in detail in an excellent
review by Robertson in 1927 (12). Both sporogenous and non-sporogenous
types are represented. In the latter group are streptococci, micrococci,
sarcinae and lactic acid bacteria.

Just prior to 1920, many laboratories doing control work on
dairy products occasionally found small, so-called “pin-point” colonies.
These were associated with pasteurized milk. Although several inves-
tigators had been working with these “pin-point” colonies, Dotterrer
(4) was the first to recognize the fact that some of these organisms
might be growing in the pasteurizer. The work on the “pin-poin ”
colonies has been well reviewed by Tanner and Harding. (13).

Taylor was apparently the first to use laboratory pasteurization

as a control measure. He reported, in 1924, that improperly sterilized

milk utensils were a source of thermoduric organisms. (l4). Labora-
tory pasteurization was also used in 1931 by Hussong and Hammer. (9).
They found no relationship between the initial counts and the pasteur-
ization efficiency. Both high and low efficiencies were found with high
count milk and also with low count samples. In some cases there was a
tendency for high pasteurization efficiency on high count milk and a low
efficiency on low count milk.

The regular use of laboratory pasteurization for control work
on a large scale was introduced by a group of workers in the United
Dairies, London, England. (2). They too, found considerable variation
in tests made periodically on various farms. They concluded that
different methods of sanitation were responsible. Contrary to the work
of Hucker (8) who found little effect of holding temperatures for the
first four hours, these workers found that a period of more than two
hours would result in a rapid increase in heat-resistant bacteria.

These and other studies were reviewed by Hileman in 1940. (6).
He also observed that several investigators found a large percentage of
the flora of the udder was heat-resistant.

For a few years prior to the ad0ption of the tryptone glucose
extract agar as a standard plating medium in the Standard Methods for
the Examination of Dairy Products, (1) there were many papers on the
effects of changing the standard agar. Typical is the report of Hileman,
Moss and Stead (7) in which they showed that there was an increase in

both the total and thermoduric counts and that the latter increased two

and one-half to five times as much as the former. A standard of
40,000 thermoduric organisms per milliliter was recommended by

Bryan, Mallmann and Turney. (3).

EXPERIMENTAL PROCEDURE

The thermoduric cultures used in this study were isolated from
plates made of routine raw milk samples which had been pasteurized
in the laboratory. To obtain a variety of types, the forty cultures
represent eighteen different producers. The distribution is shown in

the following listings;

Culture Numbers Producer
1 - 5 3
6 - 8 5
9 - 10 8

11 - 14 9
15 ll
16 12

17 - 18 15

19 - 20 16

21 - 24 18

25 - 26 21

27 - 28 37

29 - 30 38
31 34

32 - 34 35
35 31
36 32

37 - 38 33

39 - 40 32

Although various colony types were selected on each set of plates, the

40 cultures do not necessarily represent 40 different species. Preli-

minary identification at the start of the experiments indicated that
several cultures were closely related.

Inasmuch as the objective of these studies was growth rates, it
was felt that elimination of related or similar types could be accom-
plished by comparative study of the growth rates.

To be sure that the cultures were resistant to pasteurization by
the holding method, they were subjected to 1430 F. for 30 minutes
using the following technic:

Sterile milk was pre-heated in one-by-four inch test tubes and
then 0.5 ml. of a broth culture or agar slant suspension was introduced
into the tube. At the completion of the test period of 30 i 0.5 minutes,
the milk was plated out on tryptone glucose extract agar to determine
the survival of each test organism. Thermostatically controlled water
baths were used throughout which maintained the bath temperatures at
620 C. with a maximum variation of 1° C.

The results of the initial tests showed that all the cultures were
capable of surviving this type of pasteurization. Near the completion
of the studies, all the cultures were re-checked and found to have
retained their resistance over the period of eighteen months.

It was found that the heavy pellicle formation of several broth
cultures was very difficult to disperse. When these cultures were
carried on agar slants, they were removed and readily suspended. The
most satisfactory inoculum for the growth curves was obtained by

transferring a small portion of the growth to a tube of broth. This was

10

immediately shaken vigorously and one ml. removed to a standard
dilution blank containing 99 m1. of sterile, distilled water. One ml. was
then used as the inoculum for the test.

The standard dilution bottle was utilized for the incubation flask.
Sterile milk was used for the substratum so that the organisms could be
tested in their natural environment.

The customary procedures involved in determining a growth
curve were used. The incubation bottle containing about 100 ml. of
sterile milk was pre-heated (or pre-cooled) to the temperature of the
test and seeded with one ml. of the broth suspension, prepared as
above. After the inoculum had been dispersed by the standard technic
for shaking dilution bottles, the number of organisms per ml. was
determined by a plate count according to the Standard Methods for the
Examination of Dairy Products. (1).

The bottles were then placed in the incubator (or refrigerator)
at the test temperature. Plate counts were made of the samples at
varying intervals depending upon the organism and type of test. The
length of time the bottles were removed for sampling was kept at a
minimum and never exceeded five minutes.

Insofar as possible every effort was made to standardize condi-
tions and control the variable factors. Temperatures of the incubators
were recorded by either a continuously recording thermometer or a
maximum-minimum type thermometer. With the high temperature

incubator, the charts showed a variation of 5° C. The refrigerator

11

temperature was very constant with an occasional fluctuation of not
exceeding 2° C. The 37° C. incubator showed a variation of not more
than 3° C.

Agar plates were incubated for 48 hours at 37° C. before count-
ing to eliminate the effect of any initial lag periods due to drastic, but
sub-lethal treatment of the organisms in some of the tests.

All agar slants and agar plates were made with tryptone glucose
extract agar (Difco). Other media were formulated from Difco ingre—
dients. The maltose was passed through a Seitz filter and added to
Purple Agar Base (Difco) aseptically.

The various cultural and bio-chemical characteristic of the
organisms were determined. The cultures were all found to be Gram
positive, facultative anaerobes which did not ferment lactose but pro-
duced an acid butt and alkaline slant on maltose agar. A rennet curd
and reduction were present in all the litmus milk tubes. Differences
were found in their morphology and reactions in dextrose broth which

are given in Table II.

RESULTS

Because of the inability to obtain a constant inoculum when
using different cultures, it is difficult to compare one curve to another
on the basis of organisms per milliliter. Therefore, the results were

reduced to units which were common to every curve. This was done by

Table II. Variable characteristics of the organisms used in this study.

(Juhure
1N0.

QOQDQO'D 01$me

Fermentation
of Dextrose
broth

acid
acid
none
none
none

acid
none
none
none
acid

acid
none
none
none
none

acid
none
acid
none
none

none
none
none
none
none

none
acid
acid
none
none

acid
none
acid
none
none

acid
none
none
none
none

hiorphology

cocci in pairs
cocci in chains
cocci

short rods
cocci

large cocci

cocci

rods - bipolar staining
cocci

long, filamentous cells

long rods

cocci

cocci

rods - bipolar staining
short, small rods

cocci
cocci
cocci
cocci, Staphylococcus-like clumps
cocci

rods - bipolar staining
cocci

short rods - bipolar staining

A rods in short chains

short rods - bipolar staining
short, fat rods

rods
cocci

diplococci

rods

rods - filamentous
cocci in long chains
cocci

cocci
cocci
cocci
cocci in packets of 4 or 8
cocci

12

calculating a “growth coefficient” by dividing the count in organisms
per m1. at the initial sampling into the number of organisms per ml. at

any other time.

Organisms per ml. at “t” hours

Growth coeff1c1ent = Organisms per m1. atU hours

 

This would give the results in numbers of times the culture had in-
creased. Thus, a culture which increased from 120 to 12,000 organisms
per ml. would be comparable to another which might have increased
from 8000 to 800,000 organisms per ml. in the same period. It is recog-
nized that there are limitations to the system. Since all of the ratios
are dependent upon the initial count, a large error at this point is
reflected inversely throughout the entire curve. If it were applied over.
too great a range, such factors as food supply and accumulation of toxic
products would have to be considered. However, it does offer a means
for the comparative study of the growth rates of a large number of

heterogeneous cultures.

Growth Curves at 37° C.

Following the procedures previously outlined, growth curves
were run at 370 C. over a period of 12 hours. The data arranged accord-
ing to their increase at the end of the 12 hour period, are presented in
Table III. The cultures may be divided into groups according to their
rate of growth. The first five cultures are obviously capable of much

more rapid multiplication at this temperature than are the other cultures.

Table III. Growth rates of a related group of thermoduric organisms
at 37° C. expressed in growth coefficients.

Culture Growth coefficients
No.
' 0 2 4 6 12
hours hours hours hours hours
39 1 1.1 1.6 9.5 35,500
5 1 1.6 5.0 79. 18,000
2 1 3.0 44. 337. 12,000
38 l .6 2.5 9.2 6,744
37 1 .7 1.6 10.5 2,100
36 1 .9 1.7 21. 738
29 1 1.3 2.2 3.6 410
16 1 1.2 1.4 1.9 387
35 1 1.4 .9 2.2 341
30 1 1.1 2.1 5.4 337
32 1 .4 4.0 26. 333
27 1 1.4 2.0 5.2 253
21 1 1.2 2.4 4.5 172
26 1 1.4 2.4 5.8 129
7 l 1.1 1.7 2.7 128
12 1 .9 .8 2.5 126
28 1 1.2 2.2 2.6 114
17 l 2.0 2.3 11. 71
20 l 1.5 2.8 10. 66
9 1 - .5 5.0 60
22 1 1.0 1.8 2.5 49
19 l .7 1.1 1.1 45
18 l 1.8 2.5 24. 32
10 1 .8 1.1 1.5 24
40 1 .4 1.5 4.2 23
31 1 - 4.0 2.0 15
14 1 1.3 1.3 1.2 14
25 1 1.2 1.4 2.6 13
33 l 1.9 1.6 2.2 12
8 1 1.1 1.6 2.3 9
11 l .5 1.7 2.6 8
4 1 .9 - 1.5 7
3 l 1.5 1.6 .3 6
34 1 .6 1.5 1.5 6
15 1 1.0 1.4 1.4 3
13 1 1.7 1.3 1.3 3
1 1 .8 .8 .8 1

:qursoaboq'oén

13

The second group might be considered as those which increased at least
100 times but less than 1000 times in twelve hours. This would include
the next twelve cultures.

The remaining cultures fall into two groups--those exhibiting a
slow rate of growth and the last few which show essentially no growth
at this temperature. It is difficult to state exactly where this distinc-
tion should be made. Arbitrarily, a culture which increases less than
five times in a given period would be classed as “no growth.”

On the basis of this table, it is evident that a group having such
a range of growth characteristics would be likely to give diverse re-
sults depending upon the members of the group which are present.
These curves represent data which were obtained at a temperature not
ordinarily encountered in dairy practice. For that reason another

series of growth curves was carried out.

Growth Curves at 25° C.

In order to more closely approximate improper cooling and
storage, the complete series of cultures was tested at 25° C. The data
are presented in Table IV. Because of the longer lag phase and the
slower rate of growth at this temperature, the tests were extended to
24 hours to obtain a wider range in the results. Again using the classi-
fications of the first series, the cultures show the same breakdown into
groups according to their growth rates. The extremes are not as great

as those obtained at 37° C. It would be expected that some of them

Table) IV. Growth rates of a related group of thermoduric organisms
at 25 C. expressed in growth coefficients

Culture Growth Coefficients
No.
0 3 9 12 24
hours hours hours hours hours
38 l 2.1 5.2 7.4 4,680
26 1 1.1 3.1 16. 3,000
29 1 1.5 6.1 17. 2,780
30 1 1.2 2.0 3.0 2,250
32 1 1.5 2.2 10. 1,300
17 1 1.0 1.8 1.8 1,210
31 1 1.1 3.4 8.9 890
25 1 2.5 3.2 10. 660
20 l .4 1.7 1.2 620
18 l 1.2 1.2 1.2 596
16 l 1.7 2.9 6.4 573
22 1 1.1 1.3 1.6 530
21 1 .7 1.3 1.6 440
24 l 2.0 1.5 6.0 400
8 1 4.0 8.0 10. 400
13 1 1.1 2.3 10 230
37 1 1.1 11. 23 180
27 1 1.2 2.1 6.5 178
5 1 2.2 3.2 12 143
4 1 1.8 3.1 6 7 141
2 1 1.3 1.3 2 7 135
28 1 1.6 3.3 13 125
12 1 4.6 - 16. 115
7 1 .9 3.2 3.8 100
6 1 1.1 1.4 4.9 88
19 1 .7 1.0 1.0 87
1 1 .7 1.1 1.5 72
14 1 1.1 1.6 5.9 59
11 l .8 1.1 3.1 45
9 1 1.1 '1.8 6.4 40
3 1 1.4 2.4 9.2 34
39 1 .9 2.3 4.9 26
40 1 1.0 2.7 2.9 24
34 1 1.1 2.1 4.4 18
35 1 1.3 5.4 - 16
33 1 1.7 1.8 4.8 10
10 1 1.2 .6 1.6 5
36 1 .6 1.2 1.5 4

14

would grow much more rapidly at 37° C. than at 25° C. Although the
relationship of the various temperatures will be discussed later, it may
be pointed out here that the cultures do not occur in exactly the same
order in both sets of data. This change in temperature will have dif-
ferent effects on different members of the group of thermoduric
organisms--presumably due to different Optimum temperatures for
growth.

The above data indicate that the Optimum temperature for growth
of some of the organisms may be higher than 37° C. Tests were made
at higher temperatures to determine, if possible, the approximate limits

of growth.

Growth Curves at 51° - 55° c.

From the cultures tested a set of nine, based on growth rates at
37° C., was chosen to represent the different types for similar studies
at 51° - 55° C. The results are presented in Table V. At this temper-
ature the differentiation was very marked. Three of the cultures showed
only a slight increase, if any, followed by a rapid decrease. However,
the other six cultures, gave quite rapid growth. In some cases they
reproduced rapidly and reached the death phase of the growth curve by
24 hours and in others this stage was not reached until the third day.

Some of the thermoduric organisms had growth limits below 50°
C. as indicated by little or no increase in numbers during the test.

Others, however, were capable of more rapid growth at 50° — 55° C.

Table V. Growth rates of a selected group of thermoduric organisms at
51

- 55° C. expressed in growth coefficients.

Culture

No.

0
hours

13 1
38 1
16 l
20 l
19 1
15 1
4 1
12 1
9 1

3
hours

8.2

32.

21.

21.

9.7

7.7

2.6

6
hours

6,530
5,900
2,130
2,200
546
755

2.7

1.4

12
hours

20,000

6,250

1,600

2,200

5,600

2,900

1.4

24

hours

6,070

1,950

4,350

142

750

500

3
days

9,300

3,200

1,600

615

4.8

2,700

15

than at 37° C. and might even be classified as thermophiles if their
optimum were found to be in this range. Higher incubation temperatures

were indicated for this group.

Growth Curves at 61° - 65°C.

The temperature of pasteurization was selected for the next
series of studies. Eight of the cultures tested at 51° — 55° C. were
selected for this series. The incubator used was equipped with a re-
cording thermometer which indicated that the fluctuation of the air tem-
peratures was from 61° to 65° C. in regular, even cycles of about one
hour. Therefore, the temperature of the milk must have remained very
close to the 62° mark and actually approximated very closely pasteur-
ization for an extended period.

An examination of the data in Table VI shows that none of the
cultures were able to increase in numbers at this temperature since the
growth coefficients are all less than 1. It does indicate that several of
them are resistant to the temperature of pasteurization for eight hours

01‘ more.

Growth Curves at 5° C.

To determine the efiect of proper storage conditions, growth
studies were made at 5° C. for 3 days. The same set of cultures used
for the experiments at 51° - 55° C. and 61° - 650 C. was used in this
series. An examination of the data in Table VII reveals that these cul-

tures decreased in numbers with two exceptions. In these the increase

Table VI. Growth rates of a selected group of thermoduric organisms
at 61° - 65° C. expressed as growth coefficients.

Culture Growth Coefficients
NO' 0 3 8 16 30 2 3
hours hours hours hours hours days days

38 1 .06 .005 .005 0 .005 0

9 1 .007 .007 .003 .003 0 0
1 5 1 .02 .01 0 .02 0 0
20 1 .04 .01 0 0 0 0
12 l .62 .005 0 0 0 0
19 1 .09 0 .05 O 0 0
13 1 .04 0 0 0 0 0

16 1 .009 0 0 0 0 0

Table VII. Growth rates of a selected group of thermoduric organisms
at 5° C. expressed as growth coefficients.

Culture Growth Coefficients
N0° 0 5 12 30 2 3
hours hours hours hours days days
9 1 1.1 1.15 .97 .71 .65
12 1 1.1 .73 .26 .15 .12
19 l 1.12 .62 .30 .15 .07
4 1 .87 .66 .25 .20 .15
2 1 .75 .54 .27 .09 .07
15 1 .76 .50 .22 .09 .05
38 1 .79 .67 .20 .09 .05
13 1 .87 .62 .16 .11 .06
16 l .91 .26 .07 .03 .02

20 1 .55 .19 .06 .04 0

16

was very slight and of short duration. It is evident that prOper storage
of milk is an effective method of preventing the multiplication of ther-

moduric organisms.

Correlation of the Growth Curves

Examination of these sets of data bring out several facts which
are of interest. In the group of thermoduric organisms tested, it is
found that a wide variation in growth rates of the different organisms
occurred at all temperatures. In addition, each culture showed
entirely different growth patterns at different temperatures. In
Figures 1 and 2 are shown graphically the effect of temperature on the
growth rates of two different organisms at the various temperatures
tested.

A comparison of Tables III and IV shows that the order of
listing of the cultures has changed. Close inspection will reveal that
the majority have not changed from one extreme to another, but have
shifted only slightly. In a few cases there is little change. This is due
to the difference in Optimum temperatures which will cause one organ—
ism to grow more rapidly under one set of conditions whereas another
will have an increased rate under different conditions.

In general, the results show the great diversity of growth

activity encountered with this group of thermoduric organisms.

F/yure Z Gran/H7 Curt/es of Cu/I‘ure /.5’

 

 

 

 

 

 

 

 

Key
5' C'.
25' C’.
3 7 C'. — — ‘—
7 37°—55° c, —————-—-—
- 6,0-650 C, ___.__.__.
6 F-
'5!-
\'
. E
R l/
O A/
l /
4__
b /
I” ,1",
\ /
s /
——— 1
3 \ .-q-' — W K ’ / w x
i” yr >/’// / /
Q \\ x ‘ — — —— H
\ ,
. \
9,
‘1 -— \
2 ..
/,/ \
\
\
\
\
I - \
\ ,
\\ x
\
O t l L \ “A I l __J
0 2 4 6 8 l0 /2

Time in h oar s

0.

~l

L09. Organisms per ml.
6.

 

Figure 2, Growfh Curves of Culture 136

Key
5" C. ——————«
25° C'. -—
37’ C. — — -
5/'-55" C. ——
6/‘~65'C “——

 

 

l l
2 4 6
Time in hours

b

l

8

 

IO

[2

17

DISCUSSION

The data presented show that it would be impossible to make any
general statements on the behavior of the thermoduric group as a whole
because a number of different growth patterns were evident in the un—
selected group of organisms tested. However, the data show that the
growth curves at the various temperatures used in this study make
possible the division of the cultures into three general groups; namely,
(1) those whose rate of growth is very rapid, (2) the group with a
moderate rate of growth (an increase of a few hundred times), and (3)
the organisms that will reproduce slowly, if at all.

Even assuming the lack of any distinct separation of these
groups if a large number of cultures were studied, these three general
types of response would still be found.

Since it is impossible to obtain a bacteria-free milk, there is
no reason to assume that a producer will get the same type of organisms
contaminating his milk every day even though there is no difference in
the degree of cleanliness of his equipment. If a good sanitation program
is being followed, the tOtal count should be low and fairly constant.
However, this does not mean that the flora of the milk would remain the
same. The variety of contaminants is almost infinite and any one, or
several, might supply the predominating organisms for that milking. If
there is a lack of adequate sanitation, the possibilities of variation

would be increased proportionally.

18

Assuming that a thermoduric organism with a high rate of
growth at 25° C. contaminates the milk, the count would increase many
times during improper overnight storage. If the total count were
initially high, both the total and thermoduric counts would be prOpor-
tionally high when the milk reached the receiving station. On the other
hand, if the initial count were low and predominately thermoduric, the
total and thermoduric counts would be of the same order. If the ther-
moduric organisms had a low rate of growth at 25° C., the total count
might be high but the thermoduric count would remain low.

Many combinations of the various types of thermoduric organ-
isms could result. For example, in the case of Producer “A”, referred
to in Table I, it is conceivable, that one of his sources of possible con-
tamination was largely thermoduric organisms. On April 4, the source
of contamination was apparently of non-thermoduric types which were
readily removed by pasteurization.

With Producer “B”, the contamination seems to be mainly of
non-resistant bacteria. One exception to this is the sample taken
February 21. Since the trend of the thermoduric counts is low, it
might be assumed that the general flora of this farmer’s equipment and
barn are predominately non-resistant to heat. Perhaps this particular
instance was caused by poor cleaning of the equipment on the previous
day and thermoduric organisms which reproduced fairly well at 25° C.

were not removed. During storage of the equipment, considerable

19

multiplication could occur, and a high incidence of thermoduric organ-
isms in the milk would result.

The data on the 5° C. incubation show that none of the cultures
studied were capable of reproduction at a rate such that they would
materially increase in numbers during proper storage. Further, three
cultures which had been pasteurized were held at 5° C. for a period of
six weeks. There was no gross evidence of change in the milk, but
removal to room temperature for a period of 24 hours showed the usual
curd formation and evidence of normal growth.

The studies of growth rates presented in this thesis are particu-
larly important because they show that the incidence of thermoduric
organisms in a milk supply is not entirely the result of the initial con-
tamination of the milk. It is apparent that their numbers may be the
result of initial contamination coupled with multiplication during
improper storage. Because various thermoduric organisms show
different growth rates at various incubation temperatures, it cannot be
assumed that their presence necessarily indicates dirty equipment or
their absence indicates clean equipment.

These laboratory studies indicate the need of carefully con-
ducted field studies of both properly and improperly cleaned equipment
to resurvey the significance of thermoduric bacteria in milk. Such

studies are planned as a future project.

20

CONCLUSIONS

The thermoduric organisms studied showed a wide variation in
their individual rates of growth.

On the basis of the growth rates, these cultures may be classi-
fied into three groups--those showing rapid growth rates, those with
moderate rates of growth and those which show very little or no in-
crease in numbers.

The use of proper cooling is an effective means of preventing

the multiplication of organisms of the thermoduric type.

LITERATURE CITED

(1) American Public Health Association. Standard Methods for the
Examination of Dairy Products. 7th Ed. 1939.

(2) Anderson, E. B. and L. J. Meanwell. “Studies in the Bacteriology
of Low-Temperature Pasteurization. Part I. The Significance
of Heat-Resisting Organisms in Raw Milk Supplies.” J. Dairy
Res. 4: 213. 1933.

(3) Bryan, C. S., W. L. Mallmann and G. J. Turney. “Some Micro-
sc0pic Technics for Determining the Bacteriological Quality of
Milk.” Mich. State Coll. Agr. Exp. Sta. Circ. Bull. 186. 1944.

(4) Dotterrer, W. D. “Some Observations on High Counts in Milk
Freshly Pasteurized under Commercial Conditions.” Intern.
Assoc. Dairy and Milk Inspectors Ann. Rpt. _l_._2_: 204 - 214. 1923.

(5) Gaughran, E. R. L. “The Thermophilic Microorganisms.” Bact.
Rev. 11: 189. 1947.

(6) Hileman, J. L. “Thermoduric Bacteria in Pasteurized Milk. A
Review of the Literature.” J. Dairy Sci. _2_3: 1143 - 1160.
1940.

(7) Hileman, J. L., Clarence Moss and Betty Stead. “Thermoduric
Bacteria in Milk. ]I[. The Effect of Changing Agar and Tem-
perature of Incubation for Plate Counts on the Problem of Ther-
moduric Bacteria in Milk.” J. Dairy Sci. 24: 799. 1941.

(8) Hucker, G. J. “Studies on the Coccaceae. VIII. A Study of the
Cocci Resisting Pasteurization Temperatures.” N. Y. State
Agr. Exp. Sta. (Geneva) Tech. Bull. 134. 1928.

(9) Hussong, R. V. and B. W. Hammer. “The Pasteurization Efficien-
cies Secured with Milk from Individual Farms.” Iowa Agr.
Exp. Sta. Bull. 286. 1931.

(10) Mallmann, W. L. Unpublished data.
(11) Mallmann, W. L., Edgar Kivela, A. L. Bortree, Elbert Churchill and
L. H. Begeman. “The Influence of the Method of Sanitizing

Milking Machines on the Bacterial Content of Milk.” N. Y.
State Assoc. Milk Sanitarians Ann. Rpt. _2_Q: 177 - 190. 1946.

21

22

(12) Robertson, A. H. “ThermOphilic and Thermoduric Micro.-
organisms, with Special Reference to Species Isolated from
Milk. 1. Review of Literature.” N. Y. State Agr. Exp. Sta.
Bull. 130. 1927.

(13) Tanner, F. W. and H. G. Harding. “Thermophilic Bacteria from
Milk.” J. Bact. 11: 96. 1926.

(14) Taylor, G. B. “Some Observations on the Scientific Control of
Milk Plants from the Standpoint of Bacteria.” Intern. Assoc.
Dairy and Milk Inspectors Ann. Rpt. L3: 287. 1924.

m— v.
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A5313 54