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\y UNIVERSITY MICROFILMS
ANN ARBOR - MI CHI GAN
THE PENNSYLVANIA STATS COLLEGE
The Graduate School
Department of Dairy Husbandry
BACTERIOLOGICAL STUDIES ON SOME
REFRIGERATED DAIRY PRODUCTS
A Thesis
by
GEORGE HARRISON WATROUS, JR.
Submitted in partial fulfillment
of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
June, 1951
Approved:
Head, Department ofDairy Husbandry
TABLE OF CONTENTS
Page
INTRODUCTION 1
REVIEW OF LITERATURE 3
Effect of Refrigerated Storage on the Keeping Quality of Milk 3
Studies on Bacteria Which Grow at Refrigeration Temperatures 5
Sources of Psychrophilic Organisms in Dairy Products 8
Significance of Psychrophilic Organisms in Dairy Products 9
Effect of Incubation Temperature on Bacterial Plate Count
of Milk 11
NATURE OF THE PROBLEM AND OBJECTS OF THE STUDY 14
PLAN OF STUDY AND EXPERIMENTAL PROCEDURES 17
Experimental Study Number X 17
Experimental Studies Numbers II, III, and IV 20
Experimental Study Number V 23
Experimental Study Number VI 25
Experimental Study Number VII 27
Supplementary Information on Procedures Used 29
EXPERIMENTAL RESULTS 30
Experimental Study I 30
Experimental Study II 35
Experimental Study III 49
Experimental Study IV 54
Experimental Study V 53
Experimental Study VI 60
Experimental Study VII 63
SUMMARY AND DISCUSSION 65
CONCLUSIONS 70
BIBLIOGRAPHY 72
APPENDIX
ACKNOWLEDGEMENTS
The writer wishes to express his gratitude to Professor
F. J. Doan and Dr. D. V. Josephson for their counsel and
guidance throughout the course of this investigation. Credit
is also due to Roy E. Ginn, a senior in Dairy Husbandry, for
his assistance in the laboratory.
1
INTRODUCTION
The bacterial population of milk and other dairy products may be
determined by various official methods, including the standard plate
count, dye reduction tests, and the microscopic method. In addition,
various specialized technics have been developed to detect the presence
of certain specific organisms or groups of organisms. Standard Methods
for the Exnmination of Dairy Products (32—194-8) carefully delineates the
procedures for the various official methods, as well as the interpreta
tions of the results obtained. Particular emphasis is given to the
accepted technics, and to the interpretation of data.
Over the years various procedures have been suggested for the
bacteriological grading of dairy products. Most suggested procedures
have been based on research data indicating that existing and accepted
t
methods gave results of questionable accuracy. New plating media, changes
in incubation temperatures and wholly new technics have been attempted, and
in many cases proven to be more reliable indices of bacterial quality than
previously used methods.
Although the present official methods are widely accepted and have
been of inestimable value in improving the bacterial quality of both raw
and pasteurized milks, continuing research indicates the need for a
critical evaluation of those methods now in use. Although Standard
Methods (32-194.8) carefully stipulates interpretations, routine use of a
given procedure commonly leads to an over-simplified acceptance of the
value of the procedure. Furthermore, changing conditions and development
2
within the dairy industry may delimit the significance of formerly
satisfactory methods.
With increasing knowledge in the production and processing of dairy
products, and economic pressures forcing new methods of storage and less
frequent delivery of the finished products, it would seem that a critical
study should be made of some bacteriological methods now in use.
It has long been recognised that pasteurization of dairy products
enhances theirkeeping quality. However, even under mechanical refrigera
tion, microbiological development is a great cause of spoilage and monetary
loss. In the past, butter was one of the few dairy products stored for
extended periods of time; as a result considerable research has been
conducted on the role that bacteria, yeasts and molds play in the keeping
quality of this product. Unfortunately, in the case of fluid dairy
products, much is assumed but little is known of the affect of bacteria
on those products when held for extended periods at temperatures slightly
above freezing. Reference is made in most dairy textbooks to the growth
of psychrophilic bacteria in such products, but the data available con
cerning the sources, methods of detection and significance have indicated
the need for further study.
This investigation is concerned with seeking additional information
on the reliability of soma existing bacteriological procedures used on
raw and pasteurized milk and dairy products, and to study the types of
organisms developing in these products during extended storage at
refrigeration temperatures commonly employed.
3
REVIEW OF LITERATURE
Effect of Refrigerated Storage on the Keeping Quality of 1411k.
In milk held slightly above the freezing point, Pennington (22—
1908) noted that although the bacterial count was in the hundreds of
millions per milliliter, there was neither an odor nor taste to indicate
spoilage. She noted that it was not until visible putrefaction was under
way that the milk appeared objectionable. Ravenel, Hastings, and Hammer
(25— 1910) found a great increase in plate count and acidity of milk
held at 0° G.
In a study made by Palmer and McCutcheon (19— 1930), it was ob
served that in samples of pasteurized milk held at 5.6° C. for 6 hours
decreases in count occurred in 80 per cent of the samples and increases
in 13 per cent of the samples. Out of 105 samples held for 24. hours at
5.6° C., 68 per cent showed a decrease and 32 per cent an increase in
count.
Prescott, Bates and Needle (24— 1931) demonstrated that foods
refrigerated discontinuously showed increases in bacterial growth, the
increases being especially rapid at the higher refrigeration temperatures
employed. Nicholas and Anderson (17— 1942) reported on the keeping
qualities of regular pasteurized, pasteurized homogenized and raw milks
held at 4.4° C. They found that pasteurized milks could be held at this
temperature for a period of two weeks or more before spoilage would
occur. Milks that were removed from the refrigerator each day, shaken,
and allowed to stand at room temperature for one hour retained high
i
4
quality for approximately 10 days. These latter investigators based their
conclusions on the results of standard plate counts, titratable acidity,
and flavor observations, but did not take into account the psychrophilic
or coliform organisms. Since the standard plate counts at the time of
souring were very little different from those of the original fresh milk,
it would appear that a consideration of psychrophilic organisms would
have been helpful in interpreting their data.
In a study of the bacterial deterioration of milk, Mott and Mazer
(16— 1942) collected samples from vehicles on the streets of Boston and
held them at 4.4° C. Twenty-two samples of certified pasteurized milk
gave an original arithmetic average plate count of 3 per milliliter;
after 5 days the average count was 770. Twelve samples of grade - A
pasteurized milk began storage with an average plate count of 300, which
increased to 190,000 in 4 days. Twelve samples of grade - B pasteurized
milk were found to increase from an average of 6000 to 130,000 per
milliliter in 4 days. However, Dahlberg (3— 1945), reporting on a
comprehensive study in the New York metropolitan area, found that when
pasteurized milk was stored at 1.7° to 4*4° C., the standard plate
count was slightly lower after 4 days than when the milk was fresh.
In this latter study, milk samples stored at 7.2° to 10° C. showed a
slight decrease in standard plate counts after one day; however, at the
end of 4 days a significant increase was noted.
Thomas and Chandra Sekhar (33— 1946) found that pasteurized milk
had excellent keeping qualities for periods varying from 14 to 33 days
when held at 4.4° to 7.2° C.
5
Hammer (10— 194&), in summarizing the effects of temperature on
multiplication of organisms, noted that the higher the temperature at
which milk or cream is held, the more quickly the multiplication of
organisms begins and the more rapid the rate of their development.
Studies on Bacteria Which Grow at Refrigeration Temperatures.
Pennington (22-1908) found present in milk held just above the
freezing point, acid forming bacteria, others which acted on the protein,
and still other neutral types. Acid formers generally were present in
relatively smaller numbers than in milk held at higher temperatures but
liquefiers were more numerous.
In a study using litmus lactose agar and litmus lactose gelatin
as media, Ayers and Johnson (2— 1913) found that in raw milk the inert
group of bacteria was largest, followed, in order by the acid forming,
peptonizing and alkali formers. Pasteurization at 62.8° C. resulted
in an increase in the proportion of acid forming bacteria, and in de
creases in the alkali forming, peptonizing and inert groups. This latter
work was largely confirmed by Harding, Ward, and Harding (11— 1929), who
showed that during pasteurization there was a greater reduction in the
proportion of the non-acid forming bacteria than in other forms. In a
similar study, Black, Prouty, and Graham (A— 1932) studied 20 samples of
low count milk before and after pasteurization. Differentiation of
types of colonies was made by plating on Bacto nutrient caseinate agar
to which brom cresol purple was added prior to sterilization. In this
study the distribution of types of bacteria in the raw milk was; acid
forming, 26.17 per cent; proteolytic, 0.76 per cent; and inert and alkali
6
forming, 73.07 par cent. In the pasteurized milks the distribution was:
acid forming, 14.64 per cent; proteolytic, 0.62 per cent; and inert and
alkali forming, 84.74 per cent.
Thurston and Olson (35--3.933) in a study of low count raw and pas
teurized milks reported no tendency In either case for peptonizing
bacteria to overgrow other bacterial types present when the samples were
held four days at temperatures ranging from 2.2° to 13.3° C. They found
that at 11.7° to 13.3° C. alkali forming bacteria in the pasteurized
samples overgrew all other types, whereas with raw milks they increased
in percentage during the first day, remained constant for two days, and
decreased during the fourth, the largest percentage increase being made
by the acid forming group.
In a report on the effect of various pasteurizing temperatures on
cream, Powell (23— 1938) found that the numbers of peptonizing bacteria
were reduced by heat treatment at 68.3° C. and did not increase during
the storage period. A rather large number of alkali forming organisms
existed throughout the storage period in cream heated at lower tempera
tures. As the heating temperature was increased, the percentage of
peptonizers increased; this was accompanied by decreases in other groups
of organisms which were destroyed by heat treatment. The alkali forming
group was found to nearly disappear at 79.4° C.
Sherman, Cameron, and White (29— 1941) reported that the bacteria
responsible for the spoilage of milk held just above the freezing point
appeared to be mainly, if not entirely, gram negative, non-spore forming
rods largely of the Pseudomonas group. Spore forming bacteria played
