ABSTRACT
Title of Thesis: EFFECT OF ACID ADAPTATION ON THE THERMAL
INACTIVATION OF LISTERIA MONOCYTOGENES IN
HEATING MENSTRUA HAVING VARIOUS
COMBINATIONS OF PH AND WATER ACTIVITIES
Sharon G. Edelson-Mammel, Master of Science, 2003
Thesis Directed by: Professor Sam W. Joseph
Department of Cell Biology & Molecular Genetics
The impact of prior growth and food matrix conditions on thermal resistance of
L. monocytogenes, a causative agent of the foodborne illness listeriosis, was evaluated in
this study.  
In an initial study ( Main Study #1), acid induced and non-induced cells of
thirteen strains of Listeria monocytogenes were placed into two heating menstrua: pH
3.0, water activity (Aw) 0.987 and pH 7.0, Aw 0.970. In twelve out of twenty-six
combinations, non-induced cells were more heat resistant than induced cells. L.
monocytogenes strain #201, in a follow-up study ( Main Study #2) using a factorial
design to test additional combinations of water activity and pH heating menstrua, non-
acid-induced cells generally were more heat resistant than acid-induced cells although the
acid-induced cells showed greater thermal resistance in the heating menstrua having the
lowest pH values.
An increase in thermal resistance could lead to underestimation of treatments
necessary to eliminate potential contamination by L. monocytogenes.
EFFECT OF ACID ADAPTATION ON THE THERMAL INACTIVATION OF
LISTERIA MONOCYTOGENES IN HEATING MENSTRUA HAVING VARIOUS
COMBINATIONS OF PH AND WATER ACTIVITIES
by
Sharon G. Edelson-Mammel
Thesis submitted to the Faculty of the Graduate School of the 
University of Maryland, College Park in partial fulfillment
 of the requirements for the degree of 
Master of Science
2003
Advisory Committee:
Professor Sam W. Joseph, Chair
Professor George Bean
Dr. Robert L. Buchanan
Professor Jianahong Meng
Dr. Richard R. Whiting

ii
DEDICATION
I would like to dedicate this thesis to my husband Mark and my two children Samantha
Rose and Isaac Anthony. Without their support I would have never been able to
complete this project.
iii
ACKNOWLEDGEMENTS
I would like to thank all of my co-workers at the FDA for their support and assistance. I
would like to especially thank my supervisor Dick Whiting. I would like to thank my
schoolmates and teachers at UMD. My special thanks go to Dr. Meng and Dr. Bean. I
would like to give special thanks to my advisor Dr. Sam Joseph who has spent countless
hours with me. I could not have finished this without your guidance. I would also like to
thank Dr. Robert Buchanan who has been my supervisor, mentor, and friend. He helped
me start on this educational journey and has been there the entire way. And last, but
certainly not least, I would like to thank my family including my parents and siblings, and
my in-laws. I give special thanks to my husband and children.
iv
TABLE OF CONTENTS
List of Tables vi
List of Figures viii
List of Appendices x
I. Introduction 1
II. Literature Review 3
A. Characteristics of Listeria monocytogenes 3
B. Listeriosis 5
C. Treatment 8
D. Susceptible Population 8
E. Epidemiology 9
F. Infective Dose 11
G. Foods with Greater Susceptibility to Contamination 11
H. Industry Procedure for Prevention of Listeria 14
I. Food Processing and Hurdle Approach 16
J. Thermal Processing 16
K. Resistance to Thermal Processing 19
L. Molecular Mechanisms of Survival Under 
Various Types of Stresses 22
M. Acid Resistance 23
N. Acid Resistance and Increased Thermal Resistance 24
O. Resistance Modeling 25
P. Linear vs. Non-Linear Resistance Curves 27
Q. Predictive Food Microbiology 28
R. Water Activity (Aw) 28
S. Experimental Thermal Resistance Studies 29
T. Statement of the Problem 31
III. Material and Methods 34
A. Microorganisms 34
B. API Biochemical Identification 35
1. Inoculation 35
2. Interpretation 35
3. Identification 35
C. Preliminary Studies 36
1. Preliminary #1 Acid Resistance of Induced 
Cells vs Non-Induced Cells 36
2. Preliminary #2 L. monocytogenes Growth Phase 37
v
3. Preliminary #3 Thermal Study Using Log Phase 
Cells #201 in Heating Menstruum pH 3, 
Aw 0.960 38
4. Preliminary #4 pH Decline of Stationary Phase 
Cells Grown with and without Glucose 39
D. Main Studies 40
1. Heating Menstrua 40
2. Strains Utilized 41
3. Water Activity and pH of Heating Menstrua 41
4. Preparation of Cultures 42
5. Thermal Experiments of Preliminary Study #3 and 
Main Studies #1 and #2 42
6. Data Analysis 44
7. Additional Data Analysis 44
IV. Results 45
A. Microorganisms 45
B. Acid Resistance of Induced Cells vs Non-induced Cells 45
C. L. monocytogenes Growth Phase 45
D. Thermal Study Using Log phase Cells #201 in Heating
Menstruum pH 3, Aw 0.960  46
E. Decline of pH by Stationary Phase Cells of 
L. monocytogenes strain #201 grown with and without 
1% added glucose 46
F. Main Study One 47
G. Main Study Two 50
1. Mathematical Model 51
V. Discussion 53
VI. Conclusions 61
VII. Tables 63
VIII. Figures 79
IX. Appendix 99
X. Glossary 106
XI. References 107
vi
LIST OF TABLES
Table 1 Worldwide foodborne outbreaks of invasive listeriosis. 63
Table 2 Fractional factorial design used for Main Study #2. 66 
Table 3 Listeria monocytogenes strains used in the preliminary studies 
and Main Studies #1 and #2. 67
Table 4 API numerical profiles of the 13 Listeria monocytogenes 
strains studied. 68
Table 5 Starter culture counts and pH of strain #201 after 18 hr 
of incubation at 37?C. 69
Table 6 Results of various dilutions of cultures of strain #201 
inoculated into TSB with 1% glucose or without 
1% glucose to determine when cells are in the 
exponential phase of growth. 70
Table 7 Overnight and Starter Culture Counts of strain #201 
after 18 hr of incubation at 37?C in the presence or 
absence of glucose. 71
Table 8 D Value calculations of strain #201stationary growth phase 
cells in comparison to strain #201 exponential growth 
phase cells. 71
Table 9 The average pH and CFU/ml of strain #201 grown at 37?C for 
24 hr either in the presence or absence of glucose. 72
Table 10(a) The thermal resistance of 13 strains of L. monocytogenes 
grown under acidic and non-acidic conditions and 
then heated at 58?C in Main Study #1 heating 
menstruum pH 3.0, Aw 0.987. 73
Table 10(b) The thermal resistance of 13 strains of L. monocytogenes 
grown under acidic and non-acidic conditions and 
then heated at 58?C in Main Study #1 heating 
menstruum pH 7.0, Aw 0.960. 74
vii
LIST OF TABLES (CONT.)
Table 11 Thermal analysis of L. monocytogenes strain #201 grown in 
the presence of 1% glucose for 18 hr at 37?C and then 
tested  in various heating menstrua. 75
Table 12 Thermal analysis of L. monocytogenes strain #201 grown in 
the absence of 1% glucose for 18 hr at 37?C and then tested 
in various heating menstrua. 76
viii
LIST OF FIGURES
Fig 1 Photograph of heating coil apparatus used in all thermal studies 79
Fig 2 Growth of Listeria monocytogenes strain #201 on Oxoid agar 80
Fig 3 Photograph of Spiral Plater 81
Fig 4 Diagram of preliminary study #1 acid resistance of induced 82
cells vs non-induced cells of strain #201
Fig 5 Photograph of Spiral Counter 83
Fig 6 Diagram of preliminary study #2 procedure for 
L. monocytogenes Growth Phase 84
Fig 7 Diagram of procedure for Preliminary Study #3, thermal study 
using  Log phase cells #201 in heating menstruum 
pH 3.0,  Aw 0.960 85
Fig 8 Diagram of procedure for Preliminary Study #4, pH decline of 
stationary phase cells grown with and without glucose 86
Fig 9 Thermal decline model showing the time required for L. 
monocytogenes to decline in ?meat gravy? at a pH 
of 6.0 and an Aw of 0.970 87
Fig 10 Acid exposure decline curve of strain #201 grown in TSB 
either with or without either glucose or glutamine as a 
carbon source and then exposed to TSB adjusted to 
pH 2.5 using HCl for 0-6 hr 88
Fig 11 Acid exposure decline curve of strain 201 grown in TSB 
either with or without either glucose or glutamine as a 
carbon source and then exposed to TSB adjusted to 
pH 3.0 using HCl for 0-6 hr 89
Fig 12 Thermal Decline Curve of strain #204 grown either in the 
presence or absence of glucose (Glu) when placed in 
the heating menstruum at pH 3.0, Aw 0.987, 58?C 90
ix
LIST OF FIGURES (CONT.)
Fig 13 Thermal Decline Curve of strain #205 grown either in the 
presence or absence of glucose (Glu) when placed in 
the heating menstruum at pH 3.0, Aw 0.987, 58?C 91
Fig 14 Comparison of the 4D Values of the strains in the heating 
menstruum pH 3.0, Aw 0.987, 58?C 92
Fig 15 Comparison of the 4D Values of the strains in the heating 
menstruum pH 7.0, Aw 0.960, 58?C 93
Fig 16 Comparison of the T4D values of strain #201 grown in the 
presence of glucose and then exposed to 58?C in BHI 
in various combinations of pH and Aw heating menstrua 94 
Fig 17 Comparison of the T4D values of strain #201 grown in the 
absence of glucose and then exposed to 58?C in BHI 
in various combinations of pH and Aw heating menstrua 95
Fig 18 Comparison of the T4D values of strain #201 grown in the 
presence of glucose and then exposed to 58?C in MVJ
 in various combinations of pH and Aw heating menstrua 96
Fig 19 Comparison of the T4D values of strain #201 grown in the 
absence of glucose and then exposed to 58?C in MVJ 
in various combinations of pH and Aw heating menstrua 97
Fig 20 Comparison of the T4D values of strain #201 grown in the 
presence of glucose and added to various heating 
menstrua [pH (3.0 - 7.0) and Aw (0.960)] then exposed 
to 58?C in BHI with and without glucose 98
x
LIST OF APPENDICES
App A Identification by Listeria API Test 99
App B API Numerical Profiles of the thirteen Listeria monocytogenes 
strains utilized 100
App C Photographs of the API Listeria test results of ATCC strains 
showing positive and negative reactions 101
App D Standard plate counts of strain #201 grown in TSB with either 
glucose or glutamate as a carbon source with subsequent
exposure to TSB adjusted to pH 2.5 using HCl 102
App E Standard plate counts of strain #201 grown in TSB with either 
glucose or glutamate as a carbon source with subsequent
exposure to TSB adjusted to pH 3.0 using HCl 103
App F Thermal decline of strain #201 cells in logarithmic growth 
phase at 58?C followed by exposure to a heating 
menstruum of pH 3.0, Aw 0.960 104
App G  Preliminary Study #4 growth of strain #201 and pH decline. 105
1
I. INTRODUCTION
Listeria monocytogenes has been recognized as a cause of human illness for over
sixty years, but only in the last fifteen to twenty years has food been identified as its
primary means of transmission (Meng and Doyle, 1998).  L. monocytogenes is
transmitted in a variety of foods including milk, cheese, and other dairy products (Table
1).  Although it causes fewer cases of illnesses than Escherichia coli O157:H7, the
lethality rate is higher (Meng and Doyle, 1998).  It is one of the few pathogenic bacteria
that can pass the placental barrier (Doyle, et al., 1997).  Listeria spp. are also causative
agents of fish, fowl and animal listeriosis (Farber & Pagotto, 1992).
Thermal treatments are the most widely used means by which food processors
and consumers eliminate harmful microorganisms such as L. monocytogenes from foods. 
Cooking and related thermal processes are cost effective and easily applied.  However,
the extent of cooking required to achieve control of pathogenic microorganism must be
balanced against the detrimental effects of thermal processing (e.g., loss of thermal
sensitive nutrients, sensory alterations).   This is particularly important with L.
monocytogenes whose relative resistance is typically greater than other pathogens. 
While L. monocytogenes has been studied extensively over the past 20 years, there is
surprisingly little data available on the effects that prior growth conditions or pH/water
activity combinations have on the thermal resistance of the microorganism.  The
objective of the current study was to address a number of these deficiencies by (1)
developing simple systems for inducing acid adaptation in L. monocytogenes, (2)
assessing the effect of growing the microorganism in a moderately acidic environment on
2
its thermal resistance, and (3) assessing in a systematic manner the effect of various pH
and water activity combinations in heating menstrua on thermal resistance. 
3
II. LITERATURE REVIEW
A. Characteristics of Listeria monocytogenes
Listeria monocytogenes is a gram positive, facultatively anaerobic, non-spore
forming rod-shaped bacterial species within the genus Listeria.  The genus is part of the
Clostridium subbranch which includes Staphylococcus, Streptococcus, and Lactobacillus
(Doyle, 2001).  When gram stained, the rods appear as small coccobacilli (1-2 um in
length) occurring singly or in short chains. Although smooth colonies are seen on agar
plates under many conditions, rough colony characteristics were shown to emerge under
conditions of severe osmotic stress (Jorgensen, et al., 1996). When grown on blood agar
plates, the colonies are small, translucent, and gray, and form a narrow zone of beta (b)-
hemolysis. The hemolysis can be enhanced by the CAMP test in which the organism is
streaked in a line on a blood agar plate perpendicular to a streaked line of
Staphylococcus aureus.  CAMP factor secreted by Listeria produces a synergistic, bright
zone of hemolysis with the hemolysin of the S. aureus (Diagnostic Microbiology,
Koneman et al., 1992).
L. monocytogenes has peritrichous flagella which enable it to be motile in the
temperature range of 20 to 25?C. Conversely, at 37?C, it is not motile or is very weakly
motile. Tumbling motility, an important aid in clinical identification, can be seen in a wet
mount of organisms grown in a broth culture for 6 hr at 20?C.  Motility can be
demonstrated in a semisolid agar as an umbrella shaped pattern in the media after
overnight incubation at room temperature (Diagnostic Microbiology, Koneman et al.,
1992).
4
The microorganism is catalase positive, Voges-Proskaur positive, urease negative
and hydrolyzes esculin. The biochemical characteristics of L. monocytogenes include the
ability to produce acid from amygdalin, fructose, salicin, mannose, maltose, rhamnose,
and glycerol (Ryser and Marth, 1999).  Acid production is not observed with mannitol,
xylose, inositol, arabinose, and adonitol.  Listeria spp. prefer glucose as a carbohydrate
energy source (Pine et al., 1989).
L. monocytogenes is a globally dispersed, saprophytic bacterium that can be
found widely in soil, sewage and fresh water, and can grow on decaying vegetative
matter.  The organism is found in various aqueous environments such as the surface
water of canals and lakes, and freshwater tributaries (Rocourt and Cossart 1997).  It is
able to persist at low pH and low water activity (CheroutreVialette et al., 1998, Palumbo
et al., 1993), particularly at refrigeration temperatures. It can survive for long periods of
time in many environments, including survival in frozen foods for extended periods.  
Listeria monocytogenes is psychrotropic; it can grow at refrigeration
temperatures if other growth requirements are met (Stephens, et al., 1994).  However,
unlike many psychrotrophs, the bacterium has a broad temperature range; it has been
reported to grow at temperatures as low as 1 to 4?C and as high as 45?C (Stephens, et
al., 1994; Augustin, et al., 1998).  Like most mesophilic pathogens, the optimum growth
temperature for L. monocytogenes lies between 30?C and 37?C. Because of the ability of
the microorganism to grow in a broad temperature range, foods that contain Listeria and
are stored at temperatures above freezing could allow cell multiplication (Jorgensen, et
al., 1995, Smith, et al., 1991).  Listeria is more resistant than many other vegetative
5
foodborne pathogens to many of the treatments commonly used to reduce or eliminate
pathogenic bacteria.  These and other characteristics of L. monocytogenes, such as its
relatively high heat resistance, have made this foodborne pathogen difficult to control
(Hansen and Knochel, 1996, Rocourt and Cossart, 1997).
L. monocytogenes was subdivided by Seeliger in 1961 into thirteen serovars
based on O (somatic) and H (flagellar) antigens (McLauchlin, 1987).  The thirteen
serovars consist of 1/2a, 1/2b, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 based on
agglutination reactions with rabbit antisera against the O and the H antigens (Seeliger &
Hohne, 1979).  Of the 13 serovars, 95% of the human isolates fall into three of these
serovars- 1/2a, 1/2b and 4b. 
B. Listeriosis
Listeria monocytogenes is an intracellular pathogen in humans and other animals. 
Listeria monocytogenes infections in humans, which are referred to as listeriosis, can
affect the central nervous system (CNS), pregnant uterus, or bloodstream (McLauchlin,
1996).  Current data indicates a morbidity of about 2500 cases of listeriosis per year and
a mortality of 504 cases per year (Mead et al., 1999). One serovar in particular, 4b, is
responsible for 33 to 50% of sporadic human cases and a majority of the outbreaks of
listeriosis since 1981 (Rocourt and Cossart 1997).  Most often the isolates recovered
from foods belong to serovar 1/2a (Doyle et al., 2001).
In non-pregnant animals, listeriosis typically manifests either as CNS diseases
such as meningitis and encephalitis, or as septicemia. After at least 12 hours post-
6
exposure, individuals may experience gastrointestinal symptoms such as nausea,
vomiting, and diarrhea. After a few days or up to three weeks, symptoms may progress
to the more serious forms of illness.  Those at higher risk for disease are the elderly,
immunocompromised patients, and patients with AIDS.  
In pregnant women, the disease typically manifests as mild flu-like symptoms. 
However, L. monocytogenes can pass the placental barrier and spread from the mother
to the fetus, which can result in severe infections in the fetus and lead to miscarriages,
stillbirths, spontaneous abortions or the birth of a severely ill infant.  These surviving
infants may suffer from meningitis or severe neonatal septicemia (McLauchlin, 1996). 
L. monocytogenes has been recognized as a cause of human illness for over 60
years.  The first reported case of listeriosis was a World War I soldier suffering from
meningitis (Meng and Doyle, 1998, Rocourt and Cossart, 1997).  Infections caused by L.
monocytogenes are not as common as infections by other foodborne pathogens such as
Salmonella and Escherichia coli O157:H7. However, the fatality rate observed with
invasive (systemic) listeriosis (20-30%) is the highest among foodborne pathogens
(Rocourt and Cossart, 1997).  
L. monocytogenes after human consumption are internalized by resident
macrophage where they can survive and replicate.  They are then transported via blood
to regional lymph nodes, from which they can reach the liver and spleen, where most
Listeria are destroyed (Rocourt and Cossart, 1997).  Infected animals and humans are
found to have L. monocytogenes in their blood, milk, and feces and have a low potential
to infect others who come into contact with these contaminated body fluids (Donnelly
7
and Briggs, 1986).  Although exposure to L. monocytogenes is common, cases of
listeriosis are rare (Rocourt & Cossart, 1997).  A milder form of infection caused by
Listeria is listerial gastrointestinal disease.
There are 3 general steps necessary for Listeria monocytogenes to cause invasive
listeriosis:
1) exposure of the host through consumption of contaminated food.
2) invasion of the host via multiplication within and subsequent
translocation through the intestinal lining.
3) the organism is transported to the target tissue such as regional lymph
nodes, the brain, or the placenta by way of the blood.
 The organism may be dispersed throughout the body via circulating blood,
depending on the T cell response of the immune system (Rocourt & Cossart, 1997). 
Direct cell to cell spread allows the bacteria to move throughout the host and induce
formation of infection loci, while being sheltered from host defenses such as circulating
antibodies.  This mechanism allows the microorganism to gain access to targets such as
the placenta and CNS (Rocourt and Cossart, 1997, Ryser and Marth, 1999).  Survival
mechanisms of L. monocytogenes include the ability to invade and multiply in non-
phagocytic cells; to survive and multiply within macrophage; and to induce engulfment
by the host cells (Sherris, 1990).  Listeria monocytogenes infections are associated with
a set of virulence factors that enable invasion of the host cell and evasion of the host cell
phagosome.  The bacteria are able to move within the cytoplasm of the cell and spread to
neighboring cells (Gahan and Hill, 1999).  These virulence factors include internalin
8
(inlA), which is required for entry into the gastrointestinal lining; listeriolysin O (LLO)
which is used to escape from the vacuole by forming a pore; and actA which enables the
bacteria to move intracellularly by actin polymerization.
C. Treatment
Ampicillin and penicillin are the drugs of choice, but slow recovery may be
observed because of the ability of L. monocytogenes to survive within cells thus avoiding
the action of the antibiotic.  Use of bacteriostatic drugs such as chloramphenicol and
tetracycline can result in high rates of failure (Southwick and Parich,1996).
Trimethoprim-sulfamethoxazole (TMP-SMZ) which readily enters cells and kills L.
monocytogenes, may be the most effective treatment (Armstrong and Fung, 1993). 
Treatment with one part TMP to five parts SMZ for four to six weeks may be necessary
to clear the infected cells, especially in immunosuppressed patients (Armstrong, 1995). 
D. Susceptible Populations
Listeriosis is rarely a problem for healthy adults or children (McLauchlin, 1996). 
The populations most at risk for infection are the elderly, the immunocompromised, and
pregnant women.  Immunocompromised individuals most at risk are those who are
deficient in their T-cell mediated immunity (Rocourt and Cossart, 1997), for example
AIDS patients, neonates, elderly, and cancer patients (Rocourt, 1996).  Others at risk are
those who have received organ transplants and are receiving immunosuppressive therapy
(McLauchlin, 1996).  Chronic conditions can increase an individual?s susceptibility to
9
infection: therapy causing the blockage or neutralization of gastric acid that could
increase the chances of L. monocytogenes surviving passage through the stomach
(Rocourt, 1996).  Underlying conditions can increase the severity of the illness. 
Mortality can increase 38 to 40% among immunocompromised and elderly patients
(Rocourt and Cossart, 1997).  Pregnant women are themselves not at increased risk of
serious L. monocytogenes disease; however their fetuses are at risk for listeriosis,
particularly during the third trimester (Rocourt, 1996, Rocourt and Cossart, 1997). 
When the microorganism causes disease in non-pregnant women at risk, it usually
presents as meningitis or septicemia.  One of the first outbreaks due to Listeria took
place in Massachusetts in 1983,  where 49 cases of listeriosis were reported with 14
deaths (29%)(Bradshaw, et al., 1985).  Of the 49 cases, 7 patients were infants and 42
patients were immunosuppressed adults.  
E. Epidemiology
Because of the widespread distribution of L. monocytogenes,  there are numerous
opportunities for this pathogen to contaminate foods and thus pose a risk to consumers
(McLauchlin, 1996, Rocourt and Cossart, 1997).  The bacteria can easily get into a food
processing plant via soil on the animal?s carcass, on vegetative matter, or on a worker?s
hands or shoes.  Once inside a plant, the microorganism attaches readily to a variety of
surfaces and is often found in moist areas of the plant such as floor drains (Rocourt and
Cossart, 1997).  These surfaces include plastic, glass, or metal (McLauchlin, 1996). 
Although many of the outbreaks are due to cross contamination, such as transfer of the
10
microorganism from a worker?s hands or from improperly cleaned equipment to an
already cooked food product.  Listeria monocytogenes is a hardy microorganism and has
been isolated from plants, where good manufacturing practices (GMPs) were
implemented (Budo-Amoako, et al., 1992). 
One of the first documented outbreaks associated with L. monocytogenes
occurred in 1981 in Canada, where the implicated food was coleslaw (Rocourt and
Cossart, 1997).  In 1983, an outbreak in Massachusetts was suspected of being
associated with milk.  Dairy cows infected with Listeria were identified at the farm, from
which the implicated milk had been shipped.  There was no evidence that faulty
pasteurization had occurred at the dairy plant, where the milk was processed (Bunning,
et al., 1986, Donnelly, et al., 1987).  Confirmed outbreaks of listeriosis in North
America have been linked to foods of both animal and plant origin (Beuchat, et al., 1986,
Budo-Amoako, et al., 1992).  There have been several outbreaks of listeriosis associated
with dairy foods, although it was unclear if contamination of the products was due to
prior contamination and pathogen survival of the heat treatments or to post-
pasteurization contamination. (Boyle, et al., 1990, Casadei, et al., 1998).  However,
post-pasteurization recontamination is generally assumed to be the case since standard
pasteurization is sufficient to inactivate the microorganism.  A number of other
foodborne outbreaks of listeriosis have occurred in the United States and around the
world (Table 1).
11
F. Infectious Dose
The number of organisms required to cause an immune response is known as the
infective dose.  The infectious dose itself is variable and depends on three main factors:
the host, the pathogens, and the effects of the food matrix (Buchanan and Lindqvist).
The infectious dose necessary for Listeria to cause an infection depends on the
individuals and their immunologic status.  A lower dose (i.e., number of organisms)
would be sufficient for populations who are more susceptible to infections such as the
elderly and the immunocompromised (individuals with compromised T-cell immunity)
(Rocourt, 1996, Rocourt and Cossart, 1997). The pathogenicity of the microorganism
affects the infective dose, an organism with more virulence factors may require a smaller
dose for infection to occur.  
The infectious dose is also influenced by the characteristics of the food ingested. 
The type and amount of food consumed and the concentration of the pathogen in the
food affect the likelihood of infection (McLauchlin, 1996).  One potential factor is the
pH of the food consumed.  Exposure of L. monocytogenes to foods that have a
moderately acidic pH may enhance the ability of the microorganism to survive in the
acidic environment of the stomach, thereby reducing the number of organisms required
to cause an infection. 
G. Foods with Greater Susceptibility to Contamination
Foods associated with outbreaks have largely been refrigerated, processed,
ready-to-eat foods, i.e. consumed without prior cooking or reheating (Doyle, et al.,
12
1997) and that supported the growth of L. monocytogenes during refrigerated storage. 
These foods are often heat treated, thereby eliminating competing microorganisms,
which extends shelf life at refrigeration temperatures (McLauchlin, 1996, Rocourt and
Cossart, 1997).  The extended storage time affords L. monocytogenes, when present, the
opportunity to grow to high numbers.  Marginal temperature abuse (e.g. 8 to 12?C),
during refrigerated distribution and storage may accelerated the growth of the
microorganism (Beuchat, et al., 1986).  These fluctuations in refrigerated storage
temperatures have been found to commonly occur (Chawla, et al., 1996).
The ability of a food to support the growth of a microorganism is dependent on
an array of intrinsic and extrinsic characteristics.  Intrinsic factors are those that are
inherently associated with the food such as pH and water activity (Cheroutre Vialette, et
al., 1998), whereas extrinsic factors are those determined by the conditions of storage,
such as temperature or gaseous environment.  Care must be taken to consider what the
microorganism is experiencing and not the composite characteristics of the food in
evaluating the potential for a food to support the growth of L. monocytogenes.  A food
will often contain multiple micro-environments, any of which may support the growth of
bacteria present in the food (Montville, 1997).  There has been some speculation that
changes in food processing and distribution, particularly the extension of shelf life
through refrigerated storage, may have contributed to the emergence of L.
monocytogenes as a foodborne pathogen (Rocourt and Cossart 1997).  
L. monocytogenes has been found in a wide variety of retail, ready-to-eat foods
including milk and dairy products, meats (e.g., sausage, pate), fresh produce and fish (i.e,
13
finfish, bivalves, crustaceans), reflecting the increasingly cosmopolitan nature of the
marketplace (Rocourt and Cossart, 1997, Yen, et al., 1991).  In developing their L.
monocytogenes risk assessment, FDA/FSIS reviewed exposure data for 23 ready-to-eat
food categories which represented more than 400,000 samples (DHHS/USDA, 2003). 
The microorganism was isolated from the different food categories at a mean frequency
that ranged from 0.2 CFU for ice cream to 12.9 CFU for smoked seafood.  The presence
of L. monocytogenes has also been reportedfor other foods not covered by the risk
assessment such as liquid egg product, although transmission to humans from that
product has not been documented (Schuman and Sheldon, 1997).  
The risks in each category of ready-to-eat foods are not equal.  In the deli meats
category, there is more of a problem with contamination in poultry versus other meats. 
Roast beef and summer sausage appear to support the least growth of the
microorganism.  It has also been found that L. monocytogenes can strongly attach to the
surface of raw meat from which it is difficult to remove.  
With seafood, shellfish are at high risk for transmitting Listeria, because it has
been found in mollusks, fresh and frozen mussels, clams, and oysters. But the types of
seafood posing the highest risk are raw fin fish and lightly preserved fish products such
as salted, marinated, fermented or cold-smoked products (Huss et al., 2000).
Susceptible foods also include fermented sausage which relies upon the low
water activity of the preserved sausage (CheroutreVialette, et al., 1998), low pH and
competition from the commonly occurring bacteria needed to ferment the sausage to
eliminate the potential threat from bacteria such as Listeria.  The normal process of
14
fermenting and drying the sausage may prevent the growth of Listeria but may not
eliminate the organism from the finished product (Glass and Doyle, 1989).  To reduce
the risk of Listeria growth during the fermentation process of beaker sausage (uses a
glass beaker as a casing), an important step was the addition of a lactic starter culture
(Glass and Doyle 1989).  The addition of a lactic starter culture was found to reduce the
number of Listeria by 1 to 2 log10 CFU/g during the fermentation process of beaker
sausage (Glass and Doyle, 1989).
Some of the food safety problems with Listeria and other bacteria may be due to
cross contamination from workers or food contact surfaces which may re-introduce the
organism into the product after it has been processed (Dorsa, et al., 1993).   Listeria
species has also been found in processed lobster, but the contamination could have been
due to under-cooking or post-cooking contamination from the packing environment
(Budo-Amoako, et al., 1992).
Currently the FDA has a zero tolerance policy which means that ready-to-eat
food samples must be negative for L. monocytogenes in two 25g samples of the food
product (Meng and Doyle, 1998).  Measures are needed to prevent such post-
pasteurization contamination from occurring in products such as processed dairy
products (Donnelly and Briggs 1986).  
H. Industry Procedures for Prevention of Listeria Contamination
L. monocytogenes is found widely in the environment (i.e., soil, plant material,
water), where it is able to survive for long periods of time.  In addition, the
15
microorganism can survive for long periods of time in foods and in food processing
facilities.  There are many avenues for the microorganism to gain entry into the food
processing plant and thus provide a means for the consumer to be exposed to
contaminated food if the organism is not eliminated from the food product. Examples of
how the microorganism enters food processing plants are via soil on workers? clothes
and shoes, transport equipment and raw plant foods.  Animals can excrete the
microorganism and contaminated hides and hooves may be a source of contamination
(Food Microbiology Fundamentals and Frontiers, 2001).  Once in the processing plant,
the organism thrives in locations that are nutrient rich and have a high humidity, such as
floor drains, condensed and stagnant water, floors, and processing equipment (Cox et
al., 1989).
There are three separate avenues through which a processor can reduceor
eliminate the threat from L. monocytogenes. 
1) Reduce the chances of the microorganism entering the processing plant
by washing the soil off of workers? clothes and transport equipment. 
Make sure there is adequate clean up of animal excrement.
2) Identify the problem locations in plants and apply more stringent
cleaning routines and standards.
3) Post processing re-contamination is avoided by separating unclean
zones from clean zones.
A food processor using a Hazard Analysis and Critical Control Point (HACCP)
plan studies these questions (USDA/FSIS, 1999):
16
1) Knowing potential levels of L. monocytogenes on a surface material
for products - Do they fluctuate depending on supplier?
2) What do validation results tell you about the efficacy of kill steps used
in your process?
3) If your products are exposed to an environment not known to be L.
monocytogenes free, what post kill steps are employed to prevent
contamination or re-contamination?  What do records reveal about L.
monocytogenes in the facility?
4) Does the food product support the growth of L. monocytogenes?
5 ) Does finished product testing reveal microbial contamination?
I. Food Processing and Hurdle Approach
Many methods are used to enhance the flavor and texture of the food.  These
include cooking, salting, reducing pH, drying etc. These steps are also used to reduce or
eliminate the presence of a microbial population or decrease its ability to grow. This
approach, which uses a series of sublethal steps rather than one extreme step, is referred
to as the ?hurdles? technique.  The technique is a way to alter several factors to assure
that overall, growth will not occur (Fennema, 1996).
J. Thermal Processing, General 
Though thermal processing is one of the oldest methods used to process foods
(e.g., canning, pasteurization) or to eliminate pathogenic microorganisms prior to
17
consumption (i.e., cooking), it remains the most effective and widely used preservation
technology (Farkas, 1997).  It is also used to impart specific sensory characteristics to
food such as the development of flavors, aromas, or textural changes.  However,
excessive thermal processing can also lead to the development of undesirable changes
such as the development of off-flavors, changes in texture, the loss of desirable sensorial
factors and the loss of nutritional food characteristics.  The ideal thermal process is one
that is sufficient to reduce the probability that pathogenic microorganisms of concern
would survive, maximize the development of desirable organoleptic characteristics, and
minimize the development of deleterious changes in the food (Buchanan and Edelson,
1999b).
Pasteurization, one of the most well known thermal methods, consists of mild
heat treatments that inactivate enzymes and destroy pathogenic vegetative
microorganisms (e.g., non-spore forming bacteria).  The method can extend the shelf life
of a product provided that the packaging prevents re-contamination.  The inactivation of
microorganisms can be achieved using different time-temperature combinations and can
be selected to optimize the positive and negative attributes associated with thermal
processing.  For example, high temperature short time (HTST) pasteurization of milk has
largely replaced traditional pasteurization techniques because it provides effective
microbial inactivation while causing less damage to the sensory and nutritional
characteristics of the product.  Some thermally resistant microorganisms and most spore-
formers can survive the pasteurization, therefore foods should be kept at low
temperatures to prevent their growth (Farkas, 1997).  
18
Sterilization is the complete destruction of microorganisms potentially present in
the food.  This preservation method is divided into two categories: the sterilization of the
food in their final packaging and sterilization of the foods followed by aseptic transfer of
the product to sterile packages.  Appertization, also known as canning, is the sterilization
of foods by thermal means in closed containers (Farkas, 1997).  
The use of a microwave to heat treat foods is a method based on internal heat
generation.  A difficulty with this method is the lack of uniformity of the heat as it is
applied to the entire food product (Farkas, 1997).  During the microwave treatments, if a
temperature of 70?C is reached and maintained for at least 2 min., there will be a
substantial reduction in the number of L. monocytogenes.  Survival of any of the
organisms is most likely due to uneven heating (presence of cold spots in foods) during
the heat treatment. To reduce this possibility the temperature should be measured at
different locations on the food to check for cold spots (Coote, et al., 1991).   
The minimal temperature and time required to kill Salmonella is often used as the
measure of minimal thermal conditions, but the thermal resistance of L. monocytogenes
was found to be much higher than that of Salmonella (Schoeni, et al., 1991).  Current
pasteurization processes are sufficient to destroy any L. monocytogenes that may be
present in milk (Bradshaw, et al., 1985, Bradshaw, et al., 1987).  The microorganism
was incapable of surviving heating at the times and temperatures recommended by the
FDA milk pasteurization guidelines, i.e, minimum temperature of 72?C for a minimum of
15 seconds (Donnelly and Briggs, 1986). However, this requires that all parts of the milk
achieve adequate thermal treatment, and difficulties may arise with certain pasteurization
19
technologies.  For example, splashing of milk during vat pasteurization can allow cells to
remain above the main milk level, thereby allowing them to be insufficiently heated to
assure destruction.  This potential problem could be eliminated by using airspace heaters
to ensure adequate heating of every particle of milk present in a milk vat (Donnelly, et
al., 1987).  
K. Resistance to Thermal Processing
Listeria monocytogenes is generally considered to be more heat resistant than
other non-spore forming foodborne pathogens.  For example, in ground beef Listeria
was found to be four times more heat resistant than Salmonella when placed in pouches
and submerged in a water bath (Schoeni, et al., 1991). 
In general, L. monocytogenes is more thermally resistant when grown at
 elevated temperatures (e.g., 35? to 40?C) as compared to cells grown at room
temperature or below. Listeria may respond to the increases in temperature by
synthesizing a specific set of heat shock proteins that facilitate the repair of damaged
cells and protect against further injury (Jorgensen, et al., 1995).  These proteins appear
to be analogous to the proteins synthesized by Salmonella during heat or acid shock.
However, it is important to note that while the microorganism is somewhat more heat
resistant than many of the gram negative foodborne pathogens, most thermal processes
used in the production and preparation of foods appear to be sufficient to inactivate L.
monocytogenes. 
The heat resistance of a microorganism is influenced by many factors such as
20
strain variation, growth stage, growth temperature, food matrix and composition of the
growth media (Buchanan and Edelson, 1999b, Casadei, et al., 1998, Doherty, et al.,
1998, Rowan and Anderson, 1998; Golden, et al., 1988).  Environmental factors such as
treatment temperature are also important factors that influence the heat resistance of a
microorganism such as L. monocytogenes and must be considered when using heat
inactivation (Anderson, et al., 1991, Pagan, et al., 1998).  For example, in one study
there was no increase in the thermal resistance of intracellular bacteria versus bacteria in
free suspensions of milk (Bunning, et al., 1986).  Another study by Bradshaw et al.,
1987, found that the thermal tolerance of Listeria was increased if the food, in which it
was contained, was heat sterilized.  The increased thermal tolerance may have been due
to lack of competition from other bacteria or due to changes in the food matrix due to
the sterilization process.
The growth stage of a spore former is a very important factor in the thermal
resistance of a microorganism.  Many vegetative cells of spore formers are as heat
sensitive as other vegetative cells, whereas the microorganism?s spores have substantial
thermal resistant (Farkas, 1997).  Reducing the growth temperature under both
anaerobic and aerobic conditions eventually was found to decrease the thermal resistance
of L. monocytogenes strain Scott A (Smith, et al., 1991).  The decrease in the thermal
resistance of the organism may be caused by the unsaturated fatty acid content of the
organism, which increases as the growth temperature is lowered.  The increase in the
levels of unsaturated fatty acids in the phospholipids may create a more fluid, less
viscous cytoplasmic membrane (Smith, et al., 1991).  Stationary phase bacterial cells are
21
typically found to be more resistant than exponential phase cells (Buchanan and Edelson,
1999a, Buchanan and Edelson, 1999b).
Increasing the concentration of solutes such as NaCl in foods can affect the
thermal resistance of Listeria.  Yen et al., 1991 found that a 3% increase in the NaCl
concentration reduced the thermal resistance of Listeria in ground pork.  In the same
study, the addition of a mixture used to cure meat which contained NaCl, dextrose, and
sodium phosphates increased the thermal resistance of Listeria in ground pork.  The pH
of the food also influences the thermal resistance of an organism (Pagan, et al., 1998).  
Prior exposure of the bacterial cells present in different foods to sub-lethal
environmental stresses can increase bacterial thermal tolerance.  These stresses include
temperature, water activity (CheroutreVialette, et al., 1998), starvation, pH and the
atmosphere (Farkas, 1997, Lou and Yousef, 1996, Pagan, et al., 1997).  If the length of
time that the bacteria were starved was increased, the greater the increase in the bacterial
thermal resistance (Lou and Yousef, 1996).  Bacterial cells that were adapted to an
environment of increased salinity prior to receiving heat treatments showed a 10-fold
increase in their thermotolerance when heated in minced beef and a 22- fold increase
when heated in tryptic phosphate broth (Jorgensen, et al., 1996).  An increase in the
thermal tolerance of Listeria has also been seen after heat shocking the cells prior to
heating (Augustin, et al., 1998, Fedio and Jackson, 1989, Pagan, et al., 1997).  Cells are
heat shocked by being exposed to sub-lethal temperatures for a period of time.  Heat
shocking the bacterial cells can cause them to synthesize heat shock proteins (HSP),
which might help the bacterial cells to cope with stress-induced damage by promoting
22
the degradation of abnormal proteins (Farber and Pagotto, 1992, Rowan and Anderson,
1998).  These HSP may also help to protect the cells against further damage during
thermal treatment.  The amount of protection that can be provided by prior heat
shocking depends on the temperature and the duration of the heat shock.  Farber and
Brown, 1990, found that the heat resistance of L. monocytogenes was greatly increased
if the heat shock was between 1-2 hours, while Hansel and Knochel, 1996 found that
heat shocking L. monocytogenes cells for 30 minutes at 46?C was sufficient to increase
the thermal death time 2 to 4 fold.  
Under certain thermal processing conditions, microorganisms may be injured but
not killed.  In the proper environment, the organisms may repair themselves and begin to
grow and replicate (Smith and Buchanan, 1990).  The media used to test food samples
for the presence of bacteria may only enumerate uninjured cells leaving a population of
cells undetected.  In one study by Rowan and Anderson, 1998, enrichment media were
used to recover L. monocytogenes survivors from milk after high temperature, short term
(HTST) pasteurization.
L. Molecular Mechanisms of Survival Under Various Types of Stresses
Many proteins are expressed by L. monocytogenes during times of stress.  For
example, L. monocytogenes is commonly exposed to salt during food processing and
food preservation and it has been shown that during salt concentration, 40 proteins were
either repressed or induced at a higher rate by this microorganism (Duche et al., 2002).
The protein sB regulates the expression of numerous genes during stress
23
conditions and upon entry into stationary phase.  The sB acts in a role similar to rpoS in
gram negative bacteria responding to general stress.  sB also contributes to oxidative
stress resistance and carbon starvation resistance.  Heat stress was found to be partially
independent of sB in L. monocytogenes.  Acid resistance, however, was found to be sB
dependent and pH dependent (Ferreira et al., 2001).
DsigB strains (sB mutants) are less resistant to Brain Heart Infusion broth (pH
2.5) or synthetic gastric fluid (pH 2.5) than the wild type strain. The functioning sB
protein is necessary for full resistance of L. monocytogenes to lethal acid treatments (
Ferreira et al., 2003).
Proteins whose synthesis is induced by heat shock and other stress conditions
include proteases and ATP dependent chaperones.  L. monocytogenes contains genes for
Clp ATPases including ClpC, ClpP, and ClpE that are required for stress survival and
intracellular growth.  CtsR is a stress response regulon that controls a class III heat
shock gene (Nair et al., 2000).  Expression of GAD - glutamate decarboxylase activity
by L. monocytogenes is required for survival in the acidic environment of the stomach
(Cotter et al., 2001).
M. Acid Resistance
Listeria and other bacteria show an increased resistance or tolerance to acidic
conditions after prior exposure to acidic conditions.  Acid resistance, also known as acid
habituation, occurs after the organism is exposed to moderate non-lethal acid conditions. 
The bacterium is then able to withstand formerly lethal pH conditions as low as pH 2.5
24
(Buchanan and Edelson 1999a, Buchanan and Edelson 1999b, Phan-Thanh, et al., 2000). 
Acid tolerance is the enhanced survival of bacteria at a pH range of between 2.4 and 4.0
after exposure to moderate acidic conditions (Buchanan and Edelson, 1999, Gahan and
Hill, 1999 ).  Cells with increased acid resistance, also known as the acid tolerance
response (ATR), have also been shown to have increased resistance to other stresses
such as ionizing irradiation and thermal stress (Buchanan and Edelson, 1999a, Montville,
1997).  While Listeria, following acid adaptation, did show an increase in its acid
tolerance, the virulence of the pathogen was not altered (Gahan and Hill, 1999).  
During the processing of certain foods there is ample opportunity for the bacteria
to be exposed to sub-lethal acid conditions such as through the introduction of starter
cultures to produce a quick drop in the pH or due to naturally occurring acid producing
bacteria (Farber and Pagotto, 1992).
N. Acid Resistance and Increased Thermal Resistance
Cells that have been acid-adapted show an increase in their thermal resistance in
addition to increased acid resistance compared to non -acid-adapted cells.  This form of
cross-protection has been seen to occur with E. coli (Buchanan and Edelson, 1999a,
Farber & Pagotto 1992, Jorgensen, Hansen, et al. 1999).  There are conflicting opinions
on whether L. monocytogenes also show increased thermal resistance after acid-adaption
of the cells.  Phan-Thanh et al., 2000 and Mazzotta, 2001 found that L. monocytogenes 
cells which have been exposed to acidic conditions have also shown increased resistance
to other stresses such as thermal inactivation, alcohol stress, osmotic stress and
25
antimicrobial agents.  Conversely, Cole et al., 1993 found that prior stress with a low pH
led to decreased heat resistance.  
In the Mazzotta, 2001 study, four strains of L. monocytogenes were grown
overnight in Tryptic Soy broth (TSB) modified to pH 5.0 with HCl.  A composite of all
four strains were then used in thermal experiments.  The thermal resistance of the
composite cells was measured in three different fruit juices with a pH of 3.9 and TSB
modified to pH 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, and 7.0.  In the Phan-Thanh et al., 2000
study, the cells were exposed for two hours at pH 5.5 before the thermal resistance of
the cells was determined at 52?C.  In the Cole et al., 1993 study, the cells were only
exposed to the various pH?s (pH 4.24 to pH 7.0) of the heating menstruum for twenty
minutes before being injected into the heating coil. 
O. Resistance Modeling 
Mathematical models have been used for a number of years by food processors to
predict critical food safety margins (Cole, Davies, et al. 1993) and to predict the
outcome of different processing conditions on the survival of microorganisms during
thermal processing (Peleg & Cole 1998).  
Some models do not take into consideration the contribution that the heating rate
or prior stresses have on the total thermal process (Stephens, Cole, et al. 1994). 
Increased thermal tolerance as a result of prior stresses create thermal death time curves
that may contain both a shoulder and a tail section (Pagan, Condon, et al. 1997). 
Currently there are models that are more accurate than others; i.e. those that allow for
26
variability of heat sensitivity throughout a cell population (Stephens, Cole, et al. 1994). 
These models have the ability to describe non-linear thermal inactivation (Farkas 1997). 
Examples of these are logistic curves, which can be a better fit for certain types of data
because they allow for the possibility of survivors (Augustin, Carlier, et al. 1998), (Cole,
Davies, et al. 1993).  The use of a logistic curve may provide a good general model in
order to predict the death of L. monocytogenes under a wide variety of environmental
conditions during procedures such as thermal processing (Cole, Davies, et al. 1993). 
There is also the Gompertz equation which is an asymmetrical sigmoidal curve or S
curve which fits many bacterial growth curves and does not assume a constant rate of
growth (Augustin, Carlier, et al. 1998), (Chawla, Chen, et al. 1996).  The Gompertz
equation also considers more data points and therefore may provide a more accurate
estimate of thermal resistance of the microorganisms (Bhaduri, Smith, et al. 1991). 
Another non-linear model used by Doherty et al., 1998, used a Gauss-Newton curve
fitting program, where the model included an initial shoulder and two populations.  The
main part of the curve consisted of the major population, while the sub-population
comprised the tail of the curve.
Ultimately these more advanced equations and models will allow food scientists
and processors to better predict the processes needed to eliminate potential pathogen
contamination of a food product.  A good example, available from the United States
Department of Agriculture, is the Pathogen Modeling Program (PMP) (USDA/ARS,
2001), a useful tool for the food processor to estimate inactivation or elimination of
foodborne pathogens.  Models are also included on the growth and survival of several
27
food borne pathogens.
Mathematical tools can be used to estimate the effect of multiple hurdles used by
the food processor; i.e. the ability of an organism to repair itself or bacterial response to
the food ecosystem (Chawla, Chen, et al. 1996).  These tools can also be used as an aid
when designing and implementing a Hazard Analysis and Critical Control Point
(HAACP) program.  
P. Linear vs. Non-Linear Resistance Curves
Thermal inactivation curves have been typically represented by log-linear models. 
These models assume that all the organisms in a population are equally sensitive to heat
(Stephens, Cole, et al. 1994).  Another assumption is that while there may be a
distribution of heat sensitivities within a cell population, all the cells will receive the same
amount of heat.  Based on this assumption, the dose response curve at a given time and
at a given temperature will be a measure of the dose received (Cole, Davies, et al. 1993). 
Most thermal curves are plots of the number of survivors versus the thermal exposure
time.  However there have been deviations from these straight line survivor curves seen
during studies on the effects of mild processing temperature on vegetative bacterial cells
(Stephens, Cole, et al. 1994 ).  A number of studies showed deviations such as the
presence of shoulders and tails, especially during milder thermal treatments (Bhaduri,
Smith, et al. 1991, Farkas 1997, Stephens, Cole, et al. 1994).  Data analyzed from such
studies revealed the potential for the presence of heat resistant sub-populations (Doherty,
McMahon, et al. 1998).  
28
Q. Predictive Food Microbiology 
Food processors need information to better determine the death of certain
bacteria under mild heating conditions in a wide range of environmental conditions. 
Predictive food microbiology is able to provide quantitative data on the impact that
different parameters, both intrinsic and extrinsic, have on the growth and decline of
certain bacteria (Buchanan & Bagi 1997).  Unlike the canning process, today?s milder
thermal processing methods have much smaller margins of safety and therefore require
more accurate and precise predictions (Cole, Davies, et al. 1993, Stephens, Cole, et al.
1994).
R. Water Activity (Aw)
Water activity or water availability or water potential (CheroutreVialette, Lebert,
et al. 1998) is the amount of free water available in a food for potential use by a
microorganism.  The Aw is a measure of the vapor pressure of the free water in a food
over the vapor pressure of pure water (Farkas 1997), the ratio is then expressed as a
fraction (Jay, 1996).  Buchanan and Bagi, 1997, found that frequently the duration of
growth phase and lag phase increase proportionally as the Aw declines.  Decreased Aw
can also reduce the time needed to kill a bacterium at a given temperature during thermal
processing.  The Aw in a food can be altered by the addition of a humectant or by the
removal of the water vapor itself.  The primary humectant currently used in the food
industry is NaCl (Buchanan & Bagi 1997).  Dehydration is the removal of the water
vapor available for microbial growth. Gram negative bacteria typically require higher Aw
29
levels than do gram positive organisms or fungi which are less sensitive to reduced Aw
levels (Farkas 1997).  The ability to remove the water vapor from a food depends on
several factors: the moisture content and composition of the food, temperature and the
moisture content of the surrounding environment (Farkas 1997).  
S. Experimental Thermal Resistance Studies
Some of the variation in the literature regarding the thermal resistance of Listeria
may be due to experimental conditions, protocols, recovery media and methods used
(Doherty, McMahon, et al. 1998).  Experimental methods include sealed tube, test tube,
differential scanning calorimetry and a submerged coil.  Differential Scanning
Calorimetry has been used to heat the bacteria in order to determine thermal death times
and has been used to compare thermal denaturation curves (thermographs) (Anderson,
Hedges, et al. 1991).  In another study conducted by Donnelly et al., 1987, that
compared the use of test tubes and sealed tubes, survivors were consistently found when
using the test tubes, where none were found under the same conditions in the sealed
tubes (Donnelly, Briggs, et al., 1987).  An advantage of both of these methods is the
ability to use both liquid samples and small solid samples during the thermal studies.  The
submerged coil can only handle microbiological media and foods that are in the liquid
state (Buchanan & Edelson 1999).  There are a number of advantages in using the
submerged coil. Samples have a short temperature equilibration time and the variable
time intervals allow easily reproducible thermal inactivation determinations (Buchanan &
Edelson 1999, Cole & Jones 1990).  The equipment is also simple to operate and use,
30
therefore there are fewer problems due to multiple user error (Cole & Jones 1990). 
Studies on the submerged coil by Cole and Jones, 1990 have shown that there is no
carry-over from one sample to the next and there is complete separation of the sample
and the sterile water used to flush or move the sample out of the coil.  
The decimal reduction time, or D value, is the rate at a specific temperature of
the reduction of the bacterial population by 90% and is inversely related to the
destruction rate.  D value determination, which describes the death kinetics of an
organism, is usually done by plotting the log10 of the survivors versus the inactivation
time (Casadei, De Matos, et al. 1998).  The D value is then obtained by taking the
negative reciprocal of slope of the best fit line (Donnelly, Briggs, et al. 1987, Lou &
Yousef 1996, Rowan & Anderson 1998).  The rate of death is constant at any given
temperature and is independent of the initial cell culture (Farkas 1997).  A thermal death
curve, which is the D value logs versus the exposure temperature, shows the relative
resistance of the organism at different temperatures.  The slope of the thermal death
curve is expressed as Z value or the temperature needed to alter the log D value by one
unit (Casadei, De Matos, et al. 1998, Farkas 1997, Stephens, Cole, et al. 1994).  The
problem with the D value is that the number is calculated from the linear sections of the
thermal death curve, not the entire curve.  Usually the shoulder and/or the tail section of
the curve is eliminated from the calculation, this could lead to an underestimation of the
time required to reduce or eliminate the pathogen (Rowan & Anderson 1998).   
The media, either selective or non-selective, used to recover cells during a
thermal study can also determine which cells are recovered.  The correct non-selective
31
media will generally recover most of the injured and non-injured cells, while the correct
selective media will mostly recover only uninjured cells (Chawla, Chen, et al. 1996,
Smith & Buchanan 1990).  The difference in plate counts between a non-selective
medium and the selective medium will reflect the degree of injury experienced by the
organism.  In a previous study by Smith and Buchanan, 1990, modified Vogel Johnson
(MVJ) agar, used as the selective agar for Listeria, did not allow for the repair and
subsequent colony formation of injured Listeria cells.  A non-selective agar should
always be used, since this medium is more likely to reveal the total number of cells, both
injured and non-injured.  This subtle delineation is very important since injured or non-
culturable cells on selective agar have the potential to repair themselves in the food or
when ingested by susceptible human populations and could lead lead to potential health
problems (Lou & Yousef 1996, Rowan & Anderson 1998).
T. Statement of the Problem
The goal of this study was to gain a better understanding of the impact that prior
growth conditions and food matrices have on the thermotolerance of L. monocytogenes.
Previous research has been performed on the effect of increased acid resistance of L.
monocytogenes and concomitant increase in the resistance of the microorganism to
additional environmental stresses such as acid and heat (Farber and Pagotto, 1992),
(Gahan & Hill, 1999), (Mazzota, 2001).  However, there has been little research to
determine if induced acid resistance increases either the resistance or sensitivity of the
microorganism to stresses such as heat, acid, and reduced water activity under
32
concurrent well-defined conditions. 
The data obtained from such experiments would then be used to develop
improved mathematical models.  Such models could be used to aid food processors in
the design of more efficient heating systems, which could reduce the loss of the sensory
properties of the food while still ensuring a safe food product for the consumer.  The
information would also provide a better understanding of the ability of the
microorganisms to become more resistant to an environmental stress.  Overall the
information derived from these experiments will enable the food processing industry to
promote a safer and tastier food product. 
In previous experiments by other investigators, cells were either grown in a
defined pH, or subsequently exposed to a low pH for a period of time before being
thermally stressed.  In addition most cells were exposed to only moderate acidic
conditions such as pH 5.0-pH 5.5.  The effects of the heating menstruum on the thermal
tolerance of the microorganism were not usually tested adequately.  Chhabra et al.,
(2002) observed the effect of different combinations of milk fats on the resistance of the
cells during the thermal process.  Previous experiments also used different methods to
test the thermal tolerance of unheated cells, e.g. utilizing open flasks immersed into the
water bath.  The heating menstruum was placed into the flask and allowed to equilibrate
before the addition of the tested suspension of organisms (Fedio and Jackson, 1989),
(Golden et al, 1988).  Also, when composite or cocktails of several different strains were
used, the results would be skewed because the cells of the most thermal resistant
strain(s) masked the less resistant strains in the cocktail. (Budu-Amoako et al., 1992,
33
Glass and Doyle, 1989, Mazzotta, 2001).  
In this research, unlike previous experiments, where the prior growth conditions
consisted of growth in either an acidogenic or non-acidogenic media, the cells in either
medium were gradually exposed to an acidic environment.  The pH in the acidogenic
medium dropped from about pH 7.0 to a pH of about 4.25 in about 12 hr due to the
utilization of the glucose by the cells.  The cells were then allowed to remain at this low
pH for about 6 hr before the start of the thermal trials.  In Main Study #1, thirteen strains
were exposed to 58?C in two different heating menstrua.  In Main Study #2, a single
strain, #201 (F4258), was exposed to the same temperature in additional heating
menstrua.  All of the heating menstrua used in these studies were Brain Heart Infusion
broth (BHI), altered in pH using either concentrated or 1.0M HCl and water activity
(Aw) using saturated NaCl.  The heating menstruum with the cells were injected into the
submerged heating coil apparatus (Fig. 1) set at 58?C to test the thermal resistance of the
organism.  Both studies tested the thermal resistance of the microorganism under varying
combinatorial conditions of altered pH and Aw.
34
III. MATERIAL AND METHODS
A. Microorganisms
The thirteen L. monocytogenes strains used in this research were obtained from
the culture collection maintained by the FDA/CFSAN microbiology laboratories (Table
3).  Strains 200 through 209 were obtained from the stock culture collection from the
Microbial Food Safety Research Unit of the USDA/ARS Eastern Regional Research
Center (Wyndmoor, PA).  Strains 210, 211, and 212 were obtained directly from the
American Type Culture Collection (ATCC, Manassas, VA).  When the strains were
obtained, they were streaked on Oxford agar (Fig 2) and checked for typical growth.
They were then Gram stained and examined for motility and hemolysis.  After
confirmation to genus, the species was identified by API Listeria tests (bioMerieux
Vitek, Inc., Hazelwood, Missouri), CAMP test reaction, and hemolytic activity.  The
thirteen strains were used in Main Study #1, while L. monocytogenes strain #201
(F4258) was used for Main Study #2 and for the preliminary studies. 
Permanent frozen stock cultures of each strain were prepared by growing the
strains in Brain Heart Infusion broth (BHI) (Becton Dickinson Diagnostic Systems
(Difco), Sparks, MD), combining the BHI culture with glycerol at a ratio of 85:15, and
then storing at -70?C.  Working stock cultures of the strains were kept in a -20?C lab
freezer.  Cultures were activated by transferring a 1.0 ml aliquot of a -20?C working
stock culture to a test tube containing 10 mL of BHI and incubating overnight at 37?C. 
This BHI culture was then used to inoculate the overnight (O/N) cultures used for the
thermal resistance trials (see below).
35
B. API Biochemical Identification
1. Inoculation
The Listeria strains were grown on TSA blood agar plates for 24 hr at 37?C,
examined, and the hemolysis noted.  Several colonies from the TSA blood agar plate
were then transferred to the API suspension medium (demineralized water), and adjusted
to a concentration equivalent to a McFarland 7 standard.  This suspension was then used
to fill the twelve reaction tubes of the API strip. The incubation tray was covered and
incubated for 18-24 hr at 36?C.
2. Interpretation
After the tray was removed from the incubator, and the proper reagents added,
the reactions were recorded as + or ? on the results sheet, along with hemolysis.
3. Identification
Identification was made using a numerical profile.  The tests were separated into
groups of three and a value 1, 2 or 4 was assigned to each. A 4 digit profile was
determined by adding the values together to provide a code number and then was
compared to the list of Listeria spp codes that were included with the test package. The
digital code could differ slightly but still be identified as L. monocytogenes (Appendix
A).
36
C. Preliminary Studies: 
1. Preliminary Study #1 Acid Resistance of Induced Cells 
vs. Non-Induced Cells
To determine if acid-induced cells were more acid resistant than non-induced
cells, and to determine if glutamine has any effect on the acid resistance, cells were
exposed to two different acid conditions, pH 2.5 and pH 3.0. To accomplish this
purpose, strain #201 was grown in 4 different modifications of Tryptic Soy broth (TSB)
(18 hr at 37?C) emended as follows: 1% glucose, 1% glutamine (++), 1% glutamine (-
+), 1% glucose(+-), absence of glucose and glutamine (--).  Before beginning the
experiment sterile TSB was modified using HCl to provide a final pH of either 3.0 or
2.5.  The broths were then re-sterilized by passage through Nalgene filters (0.2 :m pore
size) . The solutions were then aseptically aliquoted in 10 ml portions to sterile test
tubes. The day before the experiment, labeled tubes were placed into a 37?C incubator.
Eighteen hours before the start of the experiment, 0.1 ml of strain #201 was transferred
to each of the 4 overnight cultures. The cultures were then placed into the 37?C
incubator. At the start of the experiment the overnight culture containing 1% glucose
and 1% glutamine was removed from the incubator, 0.1 ml portions were transferred to
the pH 2.5 replicate set for this overnight culture. The zero hour sample was plated on
duplicate BHIA and MVJ agar plates using the spiral plater (Spiral Biotech, Bethesda
MD) (Fig. 3). The sample hour was recorded and the remaining replicate tubes were
placed back into the incubator. The pH 3.0 tubes were removed from the incubator and
the process repeated (Fig. 4). The entire procedure was repeated with the other three
37
overnight cultures. At the 2 hr, 4 hr, and 6 hr sample times, the appropriate tube was
removed from the incubator. The 100 and 102 dilutions were then plated similarly to the
zero hour sample. All plates were held at room temperature until the end of the
experiment day. The plates were then incubated at 37?C for 48 hr. Colonies on the plates
were counted using the spiral counter (Spiral Biotech, Bethesda MD)(Fig. 5) and the
averages and log values calculated using Lotus 1-2-3.
2. Preliminary Study #2 L. monocytogenes Growth Phase
To determine the growth curve of strain #201, 0.1 ml, of an 18 hr stock culture
was transferred to two 10 ml tubes of TSB, containing either 1% glucose and 1%
glutamine or TSB containing 1% glutamine, 18 hours before the start of the experiment,
then placed into a 37?C incubator. Three different sized inocula from the two different
media were then used to study the growth curves of L. monocytogenes #201 over a 9 hr
period to determine the effect that prior growth had on the cells reaching exponential
growth phase.  Therefore the acidogenic culture (grown in the presence of 1% glucose
and glutamine) was removed from the incubator and 0.1 ml transferred to a tube
containing 9.9 ml BHI and labeled tube A, the zero time recorded and the appropriate
dilution spiral plated on BHI agar (BHIA) plates. Before returning the A tube (10-2 dil)
to the incubator, 0.1 ml of the sample was transferred to a tube containing 9.9 ml BHI
which was labeled tube E (10-4 dil), the zero time was recorded and the appropriate
dilution spiral plated on BHIA. Tube E was then placed in the 37?C incubator.  An
additional 1.0 ml of the overnight culture was then transferred to a tube containing 9.0
38
ml of BHI broth.  From this tube 0.1 ml was transferred to a tube containing 9.9 ml of
BHI labeled tube C (10-3 dil), the zero time was recorded and the appropriate dilution
was spiral plated on BHIA. The tube was then placed into the 37?C incubator.  The
entire procedure was repeated using the overnight culture grown in the TSB without
glucose except that the tubes were labeled B, F and D (Fig 6).  To achieve the growth
curves of strain #201, at the 1st, 2nd, 3rd, 4th, and 7th experimental hr, the tubes were
removed from the incubator and samples taken and spiral plated on BHIA as described
above. After the last sample hour was taken, the pHs of the cultures were recorded. All
plates were held at room temperature until the end of the experimental day and were
then placed into the 37?C incubator for 48 hr. The colonies were then counted using the
spiral counter. 
3. Preliminary #3 Thermal Study Using Log phase Cells #201 in 
Heating Menstruum pH3, Aw 0.960
Test tubes of TSB containing 1% glucose and 1% glutamine and TSB containing
1% glutamine were inoculated with 0.1ml of strain #201's 18 hr stock culture and placed
into a 37?C incubator for 21 hours before initiating the thermal study of the preliminary
experiment. Three hours before the start of the thermal study, 0.1 ml of the 18 hr
overnight culture was transferred to a fresh tube containing 9.9 ml of BHI which was
then placed into the 37?C incubator. The pH was then recorded and the appropriate
dilution of the 18 hr overnight culture was spiral plated on BHIA. At the start of the
study the three hr old culture was utilized in the thermal study (Fig. 7).  Following the
39
regular procedure (described in section D.1), a thermal study using a pH 3, Aw 0.960
heating menstruum was performed using 16 sec sampling intervals. The dilutions were
plated on BHIA and placed into the 37?C incubator.  The pH was also recorded and the
appropriate dilution was made of the 3 hr overnight culture which was spiral plated on
BHIA.  After 48 hr, the colonies on the plates were enumerated using the spiral counter.
4. Preliminary #4 pH Decline of Stationary Phase Cells Grown with 
and without Glucose
To determine the decline of the pH in TSB as the cells passed into stationary
phase over a 24 hr period, the sample was divided into 2 sets. Set 1 was sampled on day
1 and set 2 sampled on day 2, which allowed for sample times over a 24 hr time period. 
At the beginning of the experiment, the + glucose overnight culture was removed from
the incubator and 0.1 ml was transferred to each tube of the replicate + glucose set,
labeled A + Glu. The zero hour tube was removed and the sample time recorded.  The
appropriate dilution was spiral plated on duplicate BHIA plates, and the pH of the
sample then measured.  The remaining sample tubes were returned to the 37?C
incubator. Using the -glucose culture, the procedure was repeated with the replicate tube
set labeled A - Glu. The + glucose and - glucose procedure was repeated with replicate
B and C. The overnight cultures were placed into the 4?C refrigerator, and then were
used to inoculate the set two samples at the end of day one. At the appropriate sample
times of day one, the correct tube was removed, the time recorded, the sample was
plated on duplicate BHIA plates, and the pH recorded. After the last sample hour of the
40
day, the overnight cultures were removed from the refrigerator. Following the same
procedure used for set one, a replicate set of tubes were inoculated, sample time
recorded, and the zero sample was removed to plate and determine the pH (Fig 8). The
tubes were placed into the incubator and samples removed at the correct times to plate
and record the pH.  All of the plates were held until the end of the sample day.  Plates
were then incubated for 40 hours before being counted using the spiral counter.
D. Main Studies
1. Heating Menstrua:
Only two combinations of pH and Aw heating menstruum were used in Main
Study #1: Aw 0.987, pH 3.0 and Aw 0.960, pH 7.0.  In addition, the effects of the prior
growth conditions of the overnight culture (acidogenic and non-acidogenic) on the
thermal resistance of L. monocytogenes was tested. The heating menstrua for both
studies were several combinations of pH and Aw. The pH of BHI was modified using
concentrated HCl, and the water activity was altered using saturated NaCl.  
In Main Study #2, however, a fractional factorial design (Table 2) was
established to examine the effect of the water activity (0.987, 0.980, 0.970, 0.960) and
pH (3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, and 7.0) of the heating menstruum, along with
the effect of prior growth conditions of the overnight culture (acidogenic and non-
acidogenic), on the thermal resistance of L. monocytogenes.  The fractional factorial
design enables the researcher to gather a broad range of information on the effects of the
heating menstrua without having to sample each pH and Aw combination. 
41
2. Strains utilized:
All thirteen strains of L. monocytogenes were examined in Main Study #1 (Table
3).  For Main Study #2 only one of the thirteen L. monocytogenes strains, #201 (F4258),
was examined.  This strain did not show either extreme (thermal sensitivity or resistance)
in the two heating menstrua used in Main Study #1.  Therefore, #201 was an ideal
representative because it had a median response to thermal conditions after exposure to
the varying pH and Aw levels.  Further it is serotype 4b, which is most commonly
associated with foodborne outbreaks (Table 1)
3. Water Activity and pH of Heating Menstrua:
Modified BHI was used to represent the effects of the usual food matrix.
Therefore, BHI was modified to have specific water activities and pH to create two
heating menstrua as follows: Menstruum #1 was BHI acidified to pH 3.0 through the
addition of concentrated hydrochloric acid.  The sodium chloride (NaCl) content of the
BHI was unaltered, thus menstruum #1 had an aw of 0.987.  Menstruum #2 consisted of
BHI to which additional NaCl was added to achieve an aw = 0.960; the pH remained
unaltered at pH = 7.0.  The pH and water activities of the two BHI solutions were
verified using a pH meter (model 8005 by VWR Scientific) and a water activity meter
(Aqua Lab model CX-2, Decagon Devices Inc).  Both BHI preparations were then
filtered sterilized into a sterile Wheaton bottle and stored at 4?C until used as a heating
menstruum.  The BHI preparations were used within fourteen days of preparation.  The
42
two menstrua selected for Main Study #1 were at the extreme ends of the heating
menstrua that would be used for Main Study #2.
4. Preparation of Culture:
Approximately eighteen hours before beginning a heating trial, one culture tube
containing 10 ml of Tryptic Soy broth (TSB) without dextrose (Difco #0155-17-4,
Sparks, MD)+ 1% glutamine (Gibco, BRL) and a second tube containing 10 ml of TSB
+ 1% glutamine + 1% glucose were inoculated for each strain being tested.  TSB + 1%
glutamine is non-acidogenic, allowing the growth of L. monocytogenes without acid
production due to fermentation of glucose.  Conversely, TSB + 1% glutamine + 1%
glucose is acidogenic, supporting growth of the microorganism with an accompanying
decrease in pH.  For consistency, the cultures grown with glucose also contained
glutamine.  The tubes were incubated at 37?C for eighteen hours. 
5. Thermal Experiments of Preliminary Study #3 and Main Studies #1 
and #2:
The thermal resistance of the thirteen strains were measured using a submerged
heating coil apparatus (model 2, Sherwood Instruments, Lynnfield, MA)(Fig 1).  The
heating coil was equilibrated to 58?C and programmed to dispense ten samples within
the time frame of the experiment over the course of a heating trial.  The heating coil
allowed for accurate and easily reproducible results (Cole and Jones, 1990). 
43
Two fifteen mL aliquots of the heating menstruum being utilized in the Main
Studies were transferred to sterile 50 mL centrifuge tubes and allowed to equilibrate to
room temperature.  A 0.9 mL portion of a acidogenic culture of the L. monocytogenes
strain was transferred to one of the centrifuge tubes.  Immediately following a rapid
mixing, 0.4 mL was transferred to a pre-cooled (4?C), 3.6-mL dilution blank of sterile
0.1% peptone water, which served as the 0-time sample.  The remainder of the
inoculated heating menstruum was injected into the heating coil and 0.4 ml samples were
dispensed at the appropriate times into individual sterile vials.  The samples were then
transferred to pre-cooled 3.6 ml dilution blanks. As soon as the heating trial with the
acidogenic culture was completed, the second centrifuge tube of heating menstruum
being utilized was inoculated with the same strain grown under non-acidogenic
conditions. 
Samples were diluted further as necessary using 0.1% peptone water and surface
plated on duplicate Brain Heart Infusion Agar (BHIA) (Becton Dickinson Diagnostic
Systems (Difco), using a spiral plater.  All plates were incubated at 37?C for 40 hr.  The
plates were then enumerated using an automatic plate counter.  Each strain was tested
once under each of the four conditions.
44
6. Data Analysis:
The slope and lag (shoulder) time of each thermal inactivation trial was
determined by fitting the data to a 2-phase inactivation model (Buchanan and Edelson,
1996b) using the curve fitting software ABACUS (Damart, 1994).  D-values were
calculated by taking the negative reciprocal of the slope of the thermal inactivation
curve.  The time to achieve a 4 log10 inactivation (t4d) was used to provide an integrated
measure of the shoulder and exponential phases of the inactivation curve (Buchanan and
Edelson, 1999b).  The t4d value was calculated using the following equation:
t4d= tlag + 4D
where tlag is the duration of the shoulder of the thermal inactivation curve.
7. Additional Data Analysis
Additional analysis of the data was carried out in the Division of Mathematics
Department of FDA.  SAS statistical software was used to develop a second-order
polynomial of the two variables, Aw and pH to provide estimates of the t4d.
45
IV. RESULTS
A. Microorganisms
All thirteen strains utilized showed typical biological L. monocytogenes
characteristics: gram positive, tumbling motility in motility agar at 23?C, and beta
hemolytic activity.  All thirteen were confirmed as L. monocytogenes by Listeria API
tests, but due to some variation in their chemical reactions had different profiles (Table 4). 
This is a good example of strain variation within a species.
B. Acid Resistance of Induced Cells vs Non-Induced Cells
Induced cells were more acid resistant than non-induced cells. Glutamine did not
affect the resistance. At both pH 2.5 and pH 3.0 the +glucose cells showed less than one
log decline after 6 hr of exposure (Fig 10 and 11). Although at the 6 hr sampling some
injury of the cells could be observed as indicated by the lower counts obtained on the MVJ
plates, in which only uninjured cells were able to grow. Non-induced cells, those grown
without glucose, had a 4 log reduction at the 2 hr sampling after exposure to pH 2.5 and a
2 log reduction after exposure to pH 3.0. By the 4 hr sampling, cells exposed to pH 2.5
were below the level of detection (Fig 10). Cells exposed to pH 3.0 were low but still
detectable at the 4 and 6 hr samplings (Fig 11).
C. L. monocytogenes Growth Phase
 A growth period of approximately 3-5 hr was necessary for both the acid induced
cells and non-acid-induced cells to reach exponential growth phase (Table 6).  Growth
46
was minimal in all the cultures before 3 hr, but there was approximately 1.5 log increase in
growth between 3-5 hr for all six culture experimental tubes.  The growth curves of all six
cultures are shown in Fig. 10.
D. Thermal Study Using Log phase Cells F4258 in Heating
Menstruum pH3, Aw 0.960
Exponential phase cells were much less resistant to the thermal conditions cells in
the stationary phase cells. The 5D value of the logarithmic phase cells was 202.4 sec. The
average 5D value of the stationary phase cells in the same heating menstruum was 371.25
sec (Table 8).  The exposure of exponential phase cells in heating menstruum pH 3.0, Aw
0.960 to 58?C showed increased thermal sensitivity when compared to stationary phase
cells.
E. Decline of pH by Stationary Phase Cells of L. monocytogenes strain 
#201 grown with and without 1% added glucose
There was a gradual decrease in the pH over a 14 hr period of cell growth (Table
9).  Acid induced cells showed a larger decrease from about pH 7.0 to pH 4.5.  Non-
induced cells also decreased their pH, but only by about 0.5.  The pH of both overnight
cultures showed minor reductions in their pH for the remaining 10 hr of incubation.  Also
by the 10th hr, the cell counts of both cultures increased by about 3.5 logs.  By 24 hr there
were only slight increases, about 0.5 log, over the 10 hr sampling.   
47
F. Main Study One
Presented in Table 10 (a) and 10(b) are the D, tlag, and the t4D values for all of the
strains in both heating menstrua.  In the heating studies that utilized menstruum #1, eight
out of the thirteen strains grown under acidogenic conditions had higher D-values and
survived longer than cells grown under non-acidogenic conditions.  However, in
menstruum #2, the results were just the opposite with six out of the thirteen strains grown
in glucose having higher D-values than those not grown in glucose.  Fig 13 is an example
of a survivor curve where the cells grown in glucose exhibit more thermal resistance, while
Fig 12 illustrates a survivor curve where the cells grown in the absence of glucose display
more thermal resistance. All of the survivor curves for thermal menstrua #1 and #2 are in
Appendix A. 
The pH range of the overnight cultures grown in the presence of glucose, ranged
from 4.16 to 5.59 with a mean of 4.67 and standard deviation of 0.36.  For the cultures
grown in the absence of glucose, the pH range was 6.65-6.92 with a mean of 6.78 and
standard deviation of 0.06.
The effect of overnight growth in glucose was not consistent among all strains;
occasionally the D-values of cells grown without glucose were higher than that of cells
grown with glucose.  This inconsistency was also observed with strain #201, the 4b
serovar strains.
In heating menstruum #1, the acidogenic cells from strains #209 and #205
exhibited the greatest thermal resistance, while the cells of strain #207 were the most
thermal sensitive ( Table 10a and Fig 14).  Likewise, in the same heating menstruum, the
48
non-acidogenic cells of #209 displayed a similar pattern of greater thermal tolerance and
strain #207, greater thermal sensitivity (Fig 14).  In thermal menstruum #2, the acidogenic
cells from strain #207 was the most thermal tolerant of the thirteen strains, while cells
from strain #209 were the most thermal sensitive (Table 10b and Fig 15).  Similarly, strain
#207 grown in the absence of glucose was the most thermal resistant strain.  However in
the absence of glucose the most thermal sensitive strain was #210 in heating menstruum
#2 (Table 10b and Fig 15).  Overall both the acidogenic and non-acidogenic cells were
more thermal resistant and therefore had higher D-values in heating menstruum #2 when
compared to the cells in heating menstruum #1.
For all organisms grown in acidogenic media, those exposed to heating menstruum
#1 had lower t4D values, indicating lesser survival, than the organisms exposed to heating
menstruum #2.  In the non-acidogenic media, the organisms exposed to heating
menstruum #1 also illustrated lower t4D values.  Heating menstruum #2 had a less acidic
pH which reduced the environmental stress load on the cells as they were exposed to the
thermal environmental stress. There was attempt to separate the effect of the pH from the
effect of the Aw.
For each heating menstruum, analysis of variance did not reveal a difference
between the t4D mean values of the acidogenic and non-acidogenic.  With the same glucose
conditions, a difference between the t4D values of the two heating menstrua was observed
(p<0.05).
Exposure of the thirteen strains in this study to acid, achieved by overnight growth
in glucose, did not show consistent effects after thermal treatments.  In each of the two
49
menstrua, approximately one half of the strains showed increased heat tolerance after acid
exposure. Many of the strains displayed a different effect in each of the two menstrua. 
The serovar 4b strains also presented inconsistent results upon exposure to acidic
conditions.  In only three out of six replicates did the acidogenic strains show increased
thermal tolerance.  Strain number 201, a 4b serovar, did not display extreme thermal
resistance or thermal sensitivity and would therefore represent the majority of 4b strains. 
When selecting a single strain for further studies of the thermal tolerance of L.
monocytogenes, strain number 201 would be a good choice for representing the
characteristics of all L. monocytogenes species.
Variance analysis of the t4D values for acidogenic condition versus the non-
acidogenic condition used the null hypothesis suggesting that there was no difference
between the means of the two groups.  In heating menstruum #1, the mean t4D value
among the thirteen strains was 205.6 with a standard deviation (SD) of 123.6 for the
acidogenic cells and a mean of 193.3 and SD 168.7 for the non-acidogenic cells.  The
calculated F (analysis of variance) value was 0.0448 and the F distribution value was
4.2597 (a = 0.05), therefore the null hypothesis was not rejected.  In heating menstruum
#2, the mean t4D value among the strains was 1331.0, with a SD of 502.9 for the
acidogenic cells and a mean of 1192.8, with a SD 582.3 for the non-acidogenic cells. The
calculated F value was 0.4194 therefore the null hypothesis was not rejected.  In
comparing the two heating menstrua, using the acidogenic cells, the calculated F value
was 61.3982 and the F distribution value was 4.2597, therefore the null hypothesis was
50
rejected.  Comparing the two heating menstrua, but using the non-acidogenic cells, the
calculated F value was 35.3921 thus also rejecting the null hypothesis.  
G. Main Study Two
In this study using a single strain, results of the t4D values under the different
conditions are presented in Tables 11 and 12 and in Figures 16, 17, 18, and 19.  Using
cells grown overnight with glucose and plated onto BHIA, at each Aw value the t4D values
peaked as the pH rose to 6.0 - 6.5 and declined at higher pHs.  In general, as the Aw
decreased at a fixed pH, the t4D value increased.  For those grown without glucose, the
pattern was similar except that the t4D value at Aw 0.980 and pH 4.0 was 1100, which was
approximately twice the value (551) observed at Aw 0.960.  In the cells grown without
glucose and plated to BHIA, the t4D values were higher than in those with glucose when
the pH conditions were above 3.5.  Using cells grown overnight with glucose and plated
to MVJ media, the peak t4D values were seen at pH 5.5 to 6.5.  Decreasing the Aw again
increased the t4D values.  For those without glucose, the t4D values peaked in the range of
pH 6 to 7.5 and were generally higher as the Aw decreased.  Cells grown in the presence of
glucose showed higher t4D values than those grown without glucose when tested in the
low pH conditions (3 and 3.5) but at less acidic conditions, the cells grown without
glucose had higher t4D values.  The t4D values of the organisms plated to MVJ were less
than those on BHIA.  In addition for samples plated on BHIA, large lag times were often
seen (>100 sec), but not seen when plated on MVJ.  The MVJ media is only able to grow
uninjured cells.
51
Some of the most striking changes in the t4D values occurred at pH 5.5 and 6.5 at
Aw of 0.970.  The cells grown in the presence of glucose and plated to BHIA in these
conditions yielded much lower t4D values than those without glucose.  The same effect was
seen in the same conditions using MVJ media.  In addition, the MVJ plated cells showed a
larger drop in t4D value at Aw 0.980 and pH 6.0 when grown without glucose compared to
the same conditions with glucose.
In the samples grown in glucose and plated on BHIA, large lag times were usually
observed (>100 sec), but not when plated on MVJ.  In samples grown without glucose,
the only large lag times were seen on samples exposed to a pH of either 4.0 or 4.5 and
plated on to BHIA. 
In figure 20, L. monocytogenes #201 cells subjected to Aw of 0.960 at pH values
ranging from 3.0 to 7.0 showed higher resistance to thermal stress at higher pH values,
peaking at pH 6.0, grown in the absence of glucose than cells grown in the presence of
glucose.  The t4D values of the cells grown without glucose were approximately twice
those with glucose at pH > 4.0.  
1. Mathematical Model
Statistical analyses of the data were performed through the courtesy of Jung Lee
from the Division of Mathematics at the FDA to provide an equation for predicting D
values for other combinations of pH and Aw.  A second-order polynomial of the variables
pH and Aw was fitted to the D-values for each combination of media and glucose: BHIA
plus glucose, BHIA without glucose, MVJ plus glucose, and MVJ without glucose.
52
Unfortunately, when the adequacy of the fitted function was checked, the test showed
significance (p<0.05) indicating that the results must be viewed with caution.
For predicting D-values adjusted for a lag time, the equation is:
Y = 7.234 - 0.304 mda - 0.149 g - 8.404 Aw + 1.205 pH - 0.084 (pH*pH) 
with R2 (the percent of the variation about the mean accounted for by the fitted model) =
86%, where mda = 1 for the BHIA media and mda=2 for MVJ, g=1 for glucose added and
g=0 for no glucose added.  Y represents the logarithm (base 10) of the D-value, to
calculate the D-value, calculate 10Y. For predicting D-values unadjusted for lag time:
Y = 6.924 - 0.316 mda - 0.109 g - 7.716 Aw + 1.091 pH - 0.075 (pH*pH)
with R2 = 86%. From the fitted model, we can determine that the maximum D-values
within the pH range of 6.8 to 7.5 is at Aw = 0.960. The minimum D-values are at
Aw=0.987 and pH = 3.0 which is the boundary point of the experiment.  The fitted model
should not be used to predict beyond this boundary of the experimental region.
53
V. DISCUSSION
Previous studies have outlined various effects of environmental stresses on the
heat tolerance of foodborne pathogens.  For example, previous research on E. coli
O157:H7 has shown that when the organism is grown in an acidogenic environment,
thermal resistance is always greater than that for the same organism grown in a non-
acidogenic culture (Buchanan and Edelson, 1999b).  Earlier research on the thermal
resistance of Listeria has suggested that the resistance of the microorganisms is
enhanced by prior exposure to environmental stress (Donnelly et al., 1987).  The results
from this study illustrated that prior exposure to an environmental stress can alter the
thermal resistance L. monocytogenes.  Depending upon the strain tested and the
environment in which the bacteria are heated, resistance may either increase or decrease
after exposure to an acidogenic environment.
Farber and Pagotto, 1992, suggested that exposing particular strains of L.
monocytogenes to acid resulted in an initial increase in D-values.  Mazzotta, 2001a,
found that the thermal resistance of Listeria decreased as the environment became more
basic.  Taormina and Beuchat, 2001, found that exposure to an alkaline environment
increased the thermal resistance of a single strain of L. monocytogenes, serotype 4b.  In
that same study when the strain of L. monocytogenes was exposed to a chlorine
environment, thermal resistance decreased.   
Salt concentration is another factor that has been shown to affect the thermal
response of L. monocytogenes.  Increased salt concentrations used in the curing of pork
had been found to increase the thermal resistance of Listeria monocytogenes (Ryser and
54
Marth, 1999).  Another study found that osmotic shocking of the cells by different NaCl
concentrations increased the thermal resistance of Listeria (Farkas, 1997).  After a five
hour adaptation to TSB supplemented with 15% NaCl, L. monocytogenes, was found to
have increased heat resistance in surimi-based imitation crab (Mazzotta, 2001b).  Salt
concentration can also affect the organisms resistance to other stresses such as nisin, a
bacteriocin, used in the food industry as a preservative.  De Martinis et al. 1997, found
that low salt concentration (2 to 3.5%) protects L. monocytogenes against the
bactericidal effects of nisin. 
Thermal resistance may also be influenced by components present in the heating
menstruum.  These components can protect the bacterial cells from thermal damage by
stabilizing membranes or other cellular structures by stimulating the production of stress
related proteins (Rocourt and Cossart, 1997).
Preliminary studies reported herein were conducted on the acid resistance of L.
monocytogenes strain #201, (F4258) after prior exposure to acidogenic environment. 
The results from these studies indicated that prior exposure to moderate acidic
conditions increased the resistance of the microorganism to more severe acidic
conditions.  Kroll and Patchett, 1992, found that growth at a pH of 5.0 compared to pH
7.0 increased the resistance of L. monocytogenes to low pH.  Besides increasing the
ability of the microorganism to survive the acidic environment of some foods, this type
of stress response may enable the organism to better survive in the acidic environment of
the stomach or within a macrophage phagosome (O?Driscoll et al., 1996).  The increased
resistance may be due to the ability of the microorganism to increase protein synthesis
55
during the stress of acid adaptation.  Phan-Thanh and Mahouin, 1999 found that cells of
L. monocytogenes exposed to pH 5.5 for 2 hr showed increased expression of
constitutive proteins and synthesis of novel proteins.  The results from this study also
indicate that some of the strains of L. monocytogenes tested showed an acid tolerance
response (ATR).  According to Farber, 1995, ATR is enhanced survival of an
microorganism to acidic conditions (pH 2.5 to pH 4.0) after exposure to moderate acidic
conditions.  Buchanan and Edelson, 1999b, saw similar results with seven strains of
Escherichia coli O157:H7.  In that study, after cells were acid induced they became
more resistant to BHI acidified with either organic acid or HCl.  In the preliminary study
of this investigation, strain #201 also appeared to have an inducible pH dependent state
of acid resistance showing increased resistance to BHI acidified with HCl.  
Virulence may also be influenced by prior exposure to an environmental stress. 
Wiedmann et al., 1998 sequenced a region of L. monocytogenes which was identified as
the alternative sigma factor FB.  This factor has been linked with the ability of L.
monocytogenes to tolerate and survive acid stress after prior exposure to acidic
conditions (Ferreira et al., 2003).  Interestingly, Gahan and Hill, 1999 found that, while
prior exposure to acidic conditions increased the acid resistance of L. monocytogenes, it
did not increase the virulence of the microorganism.  Conversely in a study by Conte et
al., 2000, acid adapted cells of L. monocytogenes were better able to adhere to and enter
Caco-2, enterocyte-like cells, which would translate to an increased ability to pass
through the stomach and invade the intestinal tract.
56
One of the objectives of the preliminary research was to determine when the cells
of strain #201 reached the exponential growth phase.  The reduction in the pH during
exponential growth was also monitored until the cells reached the stationary growth
phase.  Exponential phase cells of strain #201 were then utilized in a thermal resistance
study.  The T4D values from this study were then compared to the T4D values of
stationary phase cells utilized in the Main Study #2 under the same thermal conditions. 
The T4D values of the exponential phase cells were almost half of the T4D values of strain
#201 stationary phase cells.  Studies by Lou and Yousef, 1996 also found that
exponential phase cells were more thermally sensitive than stationary phase cells.  On the
other hand studies conducted on L. monocytogenes by J?rgensen et al., 1999 found that
heat shocked exponential phase cells showed a greater increase in thermal resistance as
compared to heat shocked stationary phase cells.  
In Main Study #1, the influence of the growth condition on the thermal resistance
of the thirteen strains was not consistent.  In heating menstruum #1 (pH 3.0, Aw 0.987)
strain 207 was the most thermal sensitive and strain 209 was the most thermal resistant. 
But opposite observations were made in experiments with heating menstruum #2 (pH
7.0, Aw 0.960), where strain 209 was the most thermal sensitive and strain 207 was the
most thermal resistant. This provocative change described from highest to lowest
sensitivity in these two strains as the menstruum condition changes is intriguing, but
seemingly unexplainable.  In a recently published review Doyle et al, 2001, reviewed the
heat resistance of nineteen strains of L. monocytogenes tested under similar conditions.
Several strains showed thermal resistance that were more than 2.5 to 3 times greater
57
than other strains; the authors attributed these responses to strain variation.  Beuchat et
al., 1986 found that one strain of L. monocytogenes, Scott A, had increased thermal
resistance in the same heating condition versus strain LCDC 81-861.  Bradshaw et al.,
1985 also observed that one strain of L. monocytogenes had higher thermal resistance
than other strains.  The differences observed in the thermal resistance of the thirteen
strains utilized in Main Study #1, in the same heating conditions, was also most likely
due to strain variation.  This could have been due to the ability of some strains to
synthesize stress proteins, as compared with other strains.  Otherwise, the difference
noted may have been due to simple strain variation in membrane composition.  Based on
the moderate resistance and sensitivity shown by strain #201 to both heating menstrua,
the strain was selected for use in Main Study #2.
In Main Study #2, the pH of the heating menstruum had a strong effect on the
thermal tolerance of Listeria.  The t4D values in acidic heating menstrua were lower thus
indicating that cells have poorer survival in low pH, as expected.  This was also seen in a
thermal study by Beuchat et al., 1986, in which cabbage juice was used as the heating
menstruum. In the study, two strains of L. monocytogenes had increased thermal
resistance in the heating menstruum of pH 5.6 cabbage juice in comparison to the pH 4.6
cabbage juice heating menstruum. 
The water activity also showed an overall effect on the survival of the bacteria
tested.  Lower water activity conditions increased the t4D value indicating increased
survival.  The increased survival times allowed the effects of Aw to become more
apparent in the heating menstrua as they became less acidic.  In Casadei et al., 1998 the
58
thermal resistance of two strains of L. monocytogenes was higher in a heating
menstruum of butter versus a heating menstruum of tryptic soy broth.   
Glutamine-based medium vs. glucose plus glutamine-based medium provides a
simple means of studying pH-dependent stationary phase acid resistance in L.
monocytogenes.  The cells grown in the presence of glucose were then exposed to an
acidic environment created by the bacteria themselves prior to the heating trials. 
Whether plated to BHIA or MJV, the cells grown in an acidic environment showed less
heat tolerance than those grown without the presence of glucose when tested in pH of
greater than 4.0.  The prior exposure tends to help the cells survive further acidic
conditions but compared to cells grown without glucose, it did not appear to enhance the
thermotolerance of the cells in less acidic conditions.
According to Bergey?s Manual (Seelinger and Jones, 1986), catabolism of
glucose proceeds by the Embden-Meyerhof pathway both aerobically and anaerobically. 
This pathway is also referred to as glycolysis, in which glucose is catabolized to pyruvate
and is then further reduced to lactic acid (Stryer, 1995).  The lactic acid is released from
the cell, thus acidifying the tryptic soy broth environment surrounding the bacteria.  In
some of the experiments described herein, an alternate carbon source, glutamine, was
identified as a substrate that would not acidify the surrounding environment when
catabolized. Glutamine is hydrolyzed to glutamate and then catalyzed by oxidative
deamination using glutamate dehydrogenase to produce "-ketoglutarate which then
enters the tricarboxylic acid cycle to produce energy without releasing acidic products
into the environment.  
59
Listeria monocytogenes inactivation kinetics was log-linear when the cells were
grown in a non-acidogenic medium and became non-log-linear when grown in
acidogenic medium.  The t4D values of cells grown in a non-acidogenic medium were
greater than those of corresponding cells grown in acidogenic conditions medium. 
Overall thermal resistance is more complex due to non-log-linear inactivation kinetics.
Shoulders, or lag times, were not consistently seen in the analysis of the data
recorded during the thermal inactivation curves.  They were only seen when the cultures
were plated on BHIA agar, a non selective agar, thus demonstrating non-linear kinetics. 
The shoulders were not present when the cultures were plated on MVJ agar, a selective
agar.  The selective agar will allow most of the uninjured cells to grow, while not
allowing the injured cells to form colonies.  The lack of the shoulder on the curves based
on MVJ agar colony counts shows that the cells were injured but not being destroyed
initially.
The data obtained from both experiments could be utilized to enhance the
Pathogen Modeling Program.  In this program, the models are based on experimental
data obtained from many experiments.  The program is updated periodically as new data
and models are obtained.  Models included in the current program included those on
growth and thermal inactivation of L. monocytogenes.  An example of a thermal decline
curve is shown in Fig 9.  The model shows the time required for L. monocytogenes to
decline in ?meat gravy? with a pH of 6.0 and an Aw of 0.970. 
The results also indicate that further research is needed on the thermal resistance
of strain #201.  In Main Study #2, the thermal resistance of both heating menstrua Aw
60
0.960, Aw 0.970, and Aw 0.980 increased as the pH became more basic but then peaked. 
At the most basic pH values, pH 7.0 and pH 7.5, cells in both heating menstrua showed
decreases in their thermal resistance.  This was observed in cells grown in either the
presence or the absence of glucose.  But in the heating menstruum Aw 0.987 no true
peak was observed as the pH increased and became more basic.  Therefore additional
thermal studies should performed in heating menstrua with Aw 0.987 and pH values of
8.5 and 9.5 with cells grown both in the presence and absence of glucose.  
Main Study #2 should also be repeated using additional strains from Main Study
#1.  This effort would provide additional information on the effects of strain variation. 
Also fatty acid membrane analysis should be carried out on the thirteen strains to
determine if this cellular component had any role on the variation of the thermal
resistance observed.
The mathematical model provides a way of predicting bacterial survival under
varying conditions. The predicted minimum D-values occurred at Aw=0.987 and pH =
3.0 which is on the boundary of the experimental regions.  Further experimentation may
identify the stationary point of Aw and pH levels by testing pH below 3.0 and Aw above
0.987 where lower D-values may be obtained.
61
VI. CONCLUSIONS
This research showed that the growth conditions and the heating menstrua
affected the thermal resistance of L. monocytogenes.  It provides important information
on one of the most utilized methods to reduce potential pathogen contamination of food
products, i.e., thermal processing.
Although the effects of prior growth conditions were not consistent, they should
be taken into consideration, since some strains of L. monocytogenes can achieve
enhanced thermal resistance based on prior exposure to acidogenic conditions.  If not
considered, the process utilized to prepare the food product may not eliminate potential
contamination of the food product.  Therefore the food processors should ensure that the
process they utilize will eliminate the most resistant forms of the pathogen.
The effects of the heating menstrua must also have an important role in the
thermal process.  As the pH of heating menstrua became more basic, the thermal
tolerance of strain #201 increased, which would mean that the food processor would
have to increase the time a food is heated in order to eliminate the potential pathogen.
All of the findings obtained from this research will help to enhance programs such
as the Pathogen Modeling Program.  The programs are limited only by the data on which
the models are currently based.  Addition of more reliable data will enable improvements
in the accuracy of the programs.  Therefore the processor can make the thermal process
more precise for the elimination of potential contamination by Listeria monocytogenes. 
These improvements would result in reduction of the development of off-flavors, which
occur due to over-processing, thereby creating an improved food product.  While safety
62
is an important factor to the food processor, making a product that the consumers will
purchase is ultimately the raison d?etre for manufacturing the food products.
63
VII. Tables
Table 1. Worldwide foodborne outbreaks of invasive listeriosis- 
(US DHHS/USDA, 2003)a
Year Place No. of
Cases
(deaths)
Implicated (or
likely vehicle) 
% of
perinatal
cases
L.
monocytogenes
serotype
1978-
1979
Australia 12(0) raw vegetables unknown unknown
1979 Massachusetts 20 (3) (Cheese or Raw
Vegetables)
0 4b
1980 New Zealand 22(6) Raw seafood 100 1b
1981 Nova Scotia,
Canada
41 (18) Coleslaw 83 4b
1981 England 11(5) Dairy Products unknown 1/2a
1983 Massachusetts 32 (14) Pasteurized milk 22 4b
1985 California 142 (48) Mexican-style
cheese
66 4b
1983-
1987
Switzerland 122 (34) Soft cheese 53 4b
1986 Austria 28(5) Unpasteurized
milk, organic
vegetables
86 unknown
1986-
1987
Pennsylvania 36(0) Ice cream,
salami, brie
cheese
44 4b, 1/2b, 1/2a
1986-
1987
California 2(0) Raw eggs unknown 4b
1987 California 11(0) Butter unknown unknown
64
1987-
1989
England 355(94) P?t? and meat
spreads
52 4b
1989 Connecticut 10 (0) (Shrimp) 33 4b
1989-
1990
Denmark 23(0) Semi-soft cheese
(blue)
unknown 4b
not
spec.
Texas 7(0) Frozen
vegetables
43 4b
1990 Australia 11(6) P?t? and meat
spreads
100 1/2a
1991 Tasmania,
Australia
4(0) Smoked mussels 0 1/2a
1992 New Zealand 4(0) Smoked mussels 0 1/2
1992 France 279 (85) Pork tongue in
jelly
0 4b
1993 France 38(11) Rillettes 82 4b
1993 Italy 18 (0) Rice Salad 0 1/2b
1994 Illinois 48 (0) Pasteurized
chocolate milk
0 1/2b
1994-
1995
Sweden 9(2) Smoked Seafood 33 4b
1995 France 33(4) Soft Ripened
Cheese (>50%
moisture)
45 4b
1997 France 14(0) Pon l?Eveque
cheese
unknown 4b
65
1998b Multi-state,
US from a
single source
101 (21) Hot Dogs unknown 4b
1998-
1999
Finland 25(6) Butter 0 3a
1999 CT, MD, NY 11(0) P?t? 18 1/2a
1999-
2000
France 26(7) Pigs tongue in
aspic
unknown unknown
2000 10 states 29(7) Deli Turkey
meat
28 unknown
2000-
2001
North
Carolina
12(5) Mexican-style
cheese
83 unknown
2002 8 North
eastern states
63(7) Deli turkey meat 5 unknown
aCompiled from Risk assessment of the relative risk to public health from foodborne
Listeria monocytogenes among selected categories of ready-to-eat foods, USDA (86). 
66
Table 2. Fractional factorial design used for Main Study #2.
pH Aw # of Replicates
3.0 0.980 2
4.0 0.980 1
5.0 0.980 3
6.0 0.980 1
7.0 0.980 2
3.5 0.987 3
4.5 0.987 1
5.5 0.987 1
6.5 0.987 3
3.0 0.960 3
4.0 0.960 1
5.0 0.960 1
6.0 0.960 1
7.0 0.960 3
3.5 0.970 2
4.5 0.970 1
5.5 0.970 1
6.5 0.970 2
67
Table 3. Listeria monocytogenes strains used in the preliminary studies and Main Studies
#1 and #2. 
Laboratory
Strain
Number
Donor?s
Identification
Number
Serovar or
Poly O Type
(1 or 4)a
Original Source
200 Brie L 4 Brie Cheese
201 F4258 4b Clinical, CDC
202 GV6-V-S 1 Ground Veal b
203 F2-LPM-G 1 Flounderb
204 H4-VJ-G 1 Ground Beefb
205 Murray-B 4 Clinical, CDC
206 S9-NMB-S 1 Sausageb
207 ATCC 7644 1 Human Isolate 
208 MF2-L-P 1 Monk Fishb
209 RMI 4 Bovine Tissue, FDA
210 ATCC 43256 4 Mexican Style Cheese from
California 
211 ATCC 51776 4b Belgium Cheese 
212 ATCC 51414 4b Raw milk associated with an
outbreak of listeriosis in 
a Vilasini Suktankar (FDA, Atlanta, GA)
b (Buchanan et al.,1989)
68
Table 4: API numerical profiles of the 13 Listeria 
monocytogenes strains studied.
FST Strain # Profile API Profile ID
200 6410 Listeria monocytogenes
201 6510 Listeria monocytogenes
202 2510 Listeria monocytogenes
203 6450 Listeria monocytogenes
204 6510 Listeria monocytogenes
205 6450 Listeria monocytogenes
206 6110 Listeria monocytogenes
207 6450 Listeria monocytogenes
208 6450 Listeria monocytogenes
209 6510 Listeria monocytogenes
210 6510 Listeria monocytogenes
211 6510 Listeria monocytogenes
212 6410 Listeria monocytogenes
69
Table 5: Starter culture counts and pH of strain #201 after 18 hr of incubation at 37?C a.
Sample Average pH  Log [cfu/ml]
Tube 0 BHIA MVJ
with glucose and
glutamine
4.32 9.05 9.12
with glucose only    4.31 9.06 9.06
with glutamine only 7.17 8.12 8.03
without glucose
and glutamine 
7.18 8.17 7.92
aThe cells were then utilized in the preliminary study on the acid resistance of
acid induced cells vs. non acid induced cells.
70
Table 6.  Results of various dilutions of cultures of strain #201 inoculated into TSB with
1% glucose (+Glu) or without 1% glucose (w/o Glu) to determine when cells are in the
exponential phase of growtha.
Sample Tube A
(+Glu)b
Tube B
(w/oGlu)c
Tube C
(+Glu)
Tube D
(w/o Glu)
Tube Ea
(+Glu)
Tube F
(w/o Glu)
Hr (10-2
Dil?n)
(10-2
Dil?n)
(10-3
Dil?n)
(10-3
Dil?n)
(10-4
Dil?n)
(10-4
Dil?n)
0 7.38d 6.27 6.50 5.72 5.59 4.30
1 7.64 6.47 6.54 5.50 5.55 4.41
2 7.66 7.04 6.54 5.81 5.57 5.25
3 8.07 7.57 7.02 6.33 6.00 5.49
5 8.80 8.34 8.04 7.31 7.09 6.60
7 9.17 8.89 8.80 8.39 8.13 7.68
aCells grown with glucose were transferred only to tubes containing glucose.
Conversely, cells grown in the absence of glucose were transferred to tubes
without glucose.
bstrain #201 + Glu 18 hr culture count 9.01 Log (cfu/ml), Avg. pH 4.27
cstrain #201 w/o Glu 18 hr culture count 8.25 Log (cfu/ml), Avg. pH 7.15 
dResults counts are shown in [Log(cfu/ml)].
71
Table 7.  Overnight and Starter Culture Counts strain #201 after 18 hr of incubation at
37?C in the presence (+Glu) or absence (w/o Glu) of glucosea.
Sample Log [cfu/ml]  Average pH
O/N +Glu 8.61 4.35
O/N w/o Glu 8.31 7.20
3hr +Glu 8.29 6.13
3hr w/o Glu 8.02 6.30
aThe exponential phase cells were utilized in the preliminary study of the thermal
resistance of strain #201 in heating menstrum pH 3.0 Aw 0.960.
Table 8. D Value calculations of strain #201 stationary growth phase cells in comparison
to strain #201 exponential growth phase cellsa.  
Strain F4258 at different
stages of growth.
Y0 TL M RMS D
Value
5 D
8/11/00 with Glucose
(exponential  phase cells)
6.42
*
0.0* -0.02472 0.6279 40.486 202.43
7/24/00 with Glucoseb
(stationary phase cells)
7.82 197.997 -0.02180 0.2089 46.992 432.96
3/13/00 with Glucoseb
(stationary phase cells)
7.26 158.693 -0.03315 0.3397 30.167 309.53
aExperiments were performed at different times. The heating menstruum was the same
combination for each experiment - pH 3.0, Aw 0.960.
bReplicate experiments.
72
Table 9. The average pH and CFU/ml of strain #201 grown at 37?C for 24 hr either in
the presence (+Glu) or absence (w/o Glu) of glucose.a
 + Glu w/o Glu 
Time (hr) pH CFU/ml pH CFU/ml
0 7.22?0.09 5.52?0.38 7.29?0.07 5.34?0.12
2 7.09?0.00 5.97?0.04 7.14?0.02 5.89?0.08
4 7.10?0.02 6.81?0.04 7.15?0.00 6.78?0.01
6 6.56?0.01 8.11?0.02 7.09?0.01 7.96?0.06
8 6.33?0.02 8.82?0.03 6.97?0.00 8.50?0.02
9.5 5.40?0.03 8.89?0.02 6.96?0.01 8.53?0.02
14 4.43?0.02 9.11?0.09 6.97?0.01 8.70?0.03
15 4.37?0.03 9.19?0.09 6.91?0.00 8.72?0.01
16 4.34?0.01 9.37?0.03 6.93?0.01 8.69?0.05
17 4.28?0.00 9.22?0.10 6.94?0.00 8.77?0.05
18 4.24?0.01 9.31?0.04 6.94?0.00 8.75?0.12
20 4.21?0.01 9.42?0.18 6.93?0.00 8.72?0.08
22 4.18?0.00 9.41?0.08 6.95?0.03 9.00?0.06
23.5 4.14?0.03 9.33?0.05 7.00?0.01 8.71?0.16
23.75 4.14?0.01 9.41?0.00 6.96?0.00 9.09?0.00
aSee Appendix Tables 18a and 18b for original data.
73
Table 10(a). The thermal resistance of 13 strains of L. monocytogenes grown under
acidic (+ Glu) and non-acidic (w/o Glu) conditions and then heated at 58?C in Main
Study #1 heating menstruum pH 3.0 Aw 0.987a.
Strain
Number
Acidogenic (+Glu) Non-Acidogenic (w/o Glu)
Db t lagc t4Dd D t lag t4D
200 20.49 0.00 81.97 24.53 0.00 98.12e
201 28.23 0.00 112.92 45.30 20.80 202.00
202 44.90 30.81 210.41 40.52 0.00 162.08
203 41.58 0.00 166.32 23.45 15.70 109.50
204 26.32 0.00 105.28 22.74 36.74 127.70
205 76.34 108.66 414.02 62.11 0.00 248.44
206 28.30 0.00 113.20 31.75 0.00 126.98
207 12.66 0.00 50.64 11.86 0.00 47.44
208 87.80 0.00 351.20 38.79 0.00 155.16
209 79.62 103.87 422.35 178.80 13.80 729.00
210 45.14 0.00 180.56 44.19 0.00 176.76
211 64.81 0.00 259.24 31.94 14.91 142.67
212 40.67 42.07 204.75 46.88 0.00 187.52
a+ Glu mean 205.6, SD 123.6, and coefficient of variation (CV) 60.1%
w/o Glu mean 193.3, SD 168.7, and CV 87.3%
bD Value- time required for a 1 log reduction of a microorganism at a given
temperature.
cT
lag- the duration of the shoulder of the thermal inactivation curve
dT
4D- The heat process at a given temperature to reduce the population by 10^-4,equivalent to 4 times the D value + the lag time if present. 
eBold = more thermal resistant cells.
74
Table 10(b) The thermal resistance of 13 strains of L. monocytogenes grown under
acidic (+ Glu) and non-acidic (w/o Glu) conditions and then heated at 58?C in Main
Study #1 heating menstruum pH 7.0 Aw 0.960a.
Strain
Number
Acidogenic (+Glu) Non-Acidogenic (w/o Glu)
Db t lagc t4Dd D t lag t4D
200 389.11 0.00 1556.40e 181.82 0.00 727.28
201 328.69 46.94 1361.70 333.33 20.08 1353.40
202 431.03 0.00 1725.12 211.86 0.00 847.44
203 256.62 0.00 1026.48 236.02 107.49 1051.57
204 427.90 0.00 1711.60 455.27 15.78 1836.86
205 162.60 20.33 670.73 224.37 11.31 908.78
206 392.00 0.00 1568.00 201.14 119.54 924.10
207 529.35 0.00 2117.40 619.58 0.00 2478.32
208 295.12 307.24 1487.72 283.61 0.00 1134.44
209 71.35 19.76 305.16 124.71 0.00 498.84
210 364.17 45.29 1501.97 77.88 141.18 452.70
211 380.23 0.00 1520.92 384.62 0.00 1538.48
212 118.06 277.84 750.08 438.60 0.00 1754.39
a+ Glu mean 1331, SD 502.9, and coefficient of variation (CV) 37.8%
w/o Glu mean 1192.8, SD 582.3, and CV 48.8%
bD Value- time required for a 1 log reduction of a microorganism at a given
temperature.
cT
lag- the duration of the shoulder of the thermal inactivation curve
dT
4D- The heat process at a given temperature to reduce the population by 10^-4,equivalent to 4 times the D value + the lag time if present.
eBold = more thermal resistant cells.
75
Table 11. Thermal analysis of L. monocytogenes strain #201 grown in the presence of
1% glucose for 18 hr at 37?C and then tested in various heating menstrua.
Thermal
menstruuma 
Agar
medium
Time
Lag (TL)
D value (sec) 
? SD
4D value (sec) 
? SD
Aw 0.960 pH
3.0
 BHIA 178.35 36.75  ? 6.89  325.32 ? 69.33
 MVJ 50.42 34.14  ? 3.90  186.96 ? 32.33
pH
4.0
 BHIA 134.54 104.27 ? 10.02  551.61 ? 32.71
 MVJ 18.29 65.07 ? 4.60  278.56 ? 0.13
pH
5.0
 BHIA 225.29 303.49 ? 38.06 1439.25 ? 147.99
 MVJ 23.71 226.43 ? 47.12  929.44 ? 163.06
pH
6.0
 BHIA 0.00 1554.73 ? 179.6 6218.91 ? 718.37
 MVJ 0.00  787.74 ? 37.85 3150.97 ? 151.40
pH
7.0
 BHIA 465.05 868.23 ? 361.70 3937.97 ? 868.35
 MVJ 0.00 553.87 ? 80.51 2215.49 ? 322.05
Aw 0.970 pH
3.5
 BHIA 83.83 58.26 ? 26.02  316.87 ? 6.81
 MVJ 0.00 51.78 ? 2.07  207.11 ? 8.29
pH
4.5
 BHIA 251.13 104.54  669.30
 MVJ 39.67 84.67  378.35
pH
5.5
 BHIA 499.71 361.06 ? 107.41 1943.96 ? 257.49
 MVJ 91.83 370.20 ? 96.19 1572.62 ? 293.79
pH
6.5
 BHIA 0.00 812.98 ? 205.88 3251.90 ? 823.53
 MVJ 24.68 353.44 ? 60.39 1438.46 ? 241.55
pH
7.5
 BHIA 0.00 624.68  2498.72 
 MVJ NA NA NA
(cont?d)
a0.9 ml of the culture was added to 15 ml of the thermal menstruum.
76
Table 11 (cont?d). Thermal analysis of L. monocytogenes strain #201 grown in the
presence of 1% glucose for 18 hr at 37?C and then tested in various heating menstrua.
Thermal
menstruuma 
Agar
medium
Time Lag
(TL)
D value (sec) 
? SD
4D value (sec) 
? SD
Aw
0.980
pH 3.0  BHIA 123.25  46.59 ? 3.27  309.59 ? 3.42
 MVJ 14.13  29.03 ? 6.96  130.25 ? 21.75
pH 4.0  BHIA 89.93  93.24  462.87
 MVJ 0.00  67.19  268.74
pH 5.0  BHIA 153.19  276.16 ? 46.94 1257.83 ? 286.33
 MVJ 0.00  183.67 ? 34.57  734.68 ? 138.29
pH 6.0  BHIA 287.12 1086.96 ? 122.75 4347.83 ? 491.00
 MVJ 0.00 286.92 1147.67
pH 7.0  BHIA 451.58 489.24 ? 78.12 2408.52 ? 621.11
 MVJ 0.00 252.79 ? 69.61 1011.16 ? 278.43
Aw
0.987
pH 3.5  BHIA 198.04 50.23 ? 13.05 398.96 ? 75.12
 MVJ 11.49 35.91 ? 12.35 155.13 ? 33.13
pH 4.5  BHIA 178.95 109.86 ? 15.04 618.38 ? 100.71
 MVJ 51.78 50.48 ? 16.01 253.68 ? 37.69
pH 5.5  BHIA 237.97 261.67 ? 21.68 1284.65 ? 361.58
 MVJ 71.52 117.91 ? 0.86 543.16 ? 97.70
pH 6.5  BHIA 508.67 323.22 ? 89.89 1801.55 ? 160.07
 MVJ 18.83 203.96 ? 97.39 834.66 ? 371.44
pH 7.5  BHIA 326.53 612.03 2774.63
 MVJ NA NA NA
a0.9 ml of the culture was added to 15 ml of the thermal menstruum.
77
Table 12. Thermal analysis of L. monocytogenes strain #201 grown in the absence of 1%
glucose for 18hr at 37?C and then tested in various heating menstrua.
Thermal
menstruuma 
Agar
medium
Time Lag
(TL)
D value (sec) 
? SD
4D value (sec) 
? SD
Aw 0.960 pH
3.0
 BHIA 9.41 47.27 ? 6.12 198.48 ? 35.85
 MVJ 0.00 30.98 ? 6.27 123.91 ? 25.10
pH
4.0
 BHIA 100.24 112.69 ? 20.54 551.00 ? 7.67
 MVJ 0.00 84.26 ? 13.17 337.04 ? 52.70
pH
5.0
 BHIA 0.00 763.65 ? 28.20 3054.60 ? 112.80
 MVJ 67.50 180.02 ? 45.83 787.57 ? 250.84
pH
6.0
 BHIA 0.00 3061.85?1223.76 12247.4?7895.05
 MVJ 0.00 670.47 ? 72.16 2681.86 ? 288.64
pH
7.0
 BHIA 0.00 1758.71 ? 416.79 7034.82?1667.15
 MVJ 0.00 632.26 ? 40.61 2529.03 ? 162.44
Aw 0.970 pH
3.5
 BHIA 14.64 49.70 ? 8.56  213.43 ? 57.22
 MVJ 0.00 33.04 ? 14.27  132.15 ? 57.18
pH
4.5
 BHIA 231.14 111.56 ? 10.28  677.40 ? 318.16
 MVJ 0.00 65.49 ? 46.48  261.98 ? 184.38
pH
5.5
 BHIA 0.00 1731.60 ? 471.20 6926.41?1884.78
 MVJ 0.00 594.35 ? 268.56 2377.41?1074.26
pH
6.5
 BHIA 0.00 2246.18 ? 733.29 8984.73?2933.18
 MVJ 0.00 598.53 ? 5.04  2394.13 ? 20.14
pH
7.5
 BHIA 0.00 1001.60  4006.41
 MVJ NA NA NA
(cont?d)
a0.9 ml of the culture was added to 15 ml of the thermal menstruum.
78
Table 12 (cont?d). Thermal analysis of L. monocytogenes strain #201 grown in the
absence of 1% glucose for 18hr at 37?C and then tested in various heating menstrua.
Thermal
menstruuma 
Agar
medium
Time Lag
(TL)
D value (sec) 
? SD
4D value (sec) 
? SD
Aw
0.980
pH 3.0  BHIA 10.823 38.21 ? 15.014  163.65 ? 71.91
 MVJ 0.00 19.73 ? 11.78  78.90 ? 47.10
pH 4.0  BHIA 149.26 237.61  1099.69
 MVJ 3.835 88.50  357.82
pH 5.0  BHIA 0.00 665.34 ? 171.45 2661.34 ? 658.84
 MVJ 1.94 256.33 ? 55.69 1027.25 ? 219.43
pH 6.0  BHIA 0.00 1385.04 ? 179.60 5540.17 ? 718.39
 MVJ 0.00 274.37  1097.46
pH 7.0  BHIA 0.00 1124.35 ? 42.70 4497.41 ? 170.82
 MVJ 0.00 402.28 1609.13
Aw
0.987
pH 3.5  BHIA 4.22 64.66 ? 3.84 262.86 ? 10.42
 MVJ 0.00 36.63 ? 2.08 146.51 ? 8.31
pH 4.5  BHIA 214.26 162.98 ? 14.34 866.18 ? 72.04
 MVJ 0.00 78.08 ? 17.19 312.31 ? 68.76
pH 5.5  BHIA 0.00 920.56 ? 236.75 3682.22 ? 947.01
 MVJ 0.00 301.08 ? 13.86 1204.33 ? 762.53
pH 6.5  BHIA 0.00 992.06 ? 482.66 3968.25?1930.64
 MVJ 0.00 371.06 ? 118.17 1484.23 ? 472.66
pH 7.5  BHIA 0.00 1021.03 4084.13
 MVJ NA NA NA
a0.9 ml of the culture was added to 15 ml of the thermal menstruum.
79
VIII. Figures
Figure 1. Photograph of heating coil apparatus used in all thermal studies.
80
Figure 2. Growth of Listeria monocytogenes strain #201 on Oxoid agar
81
Figure 3. Photograph of Spiral Plater (Spiral Biotech, Bethesda, MD)
82
Figure 4. Diagram of preliminary study #1 acid resistance of induced cells vs non-induced
cells of strain #201
83
Figure 5. Photograph of Spiral Counter (Spiral Biotech, Bethesda, MD)
84
Figure 6.  Diagram of preliminary study #2 procedure for L. monocytogenes  growth
phase.
85
Figure 7.  Diagram of procedure Preliminary #3 thermal study using log phase cells #201
in heating menstruum pH3, Aw 0.960
86
Figure 8.  Diagram of procedure Preliminary #4 pH decline of stationary phase cells grown
with and without glucose.
87
Figure 9. Thermal decline model showing the time required for L. monocytogenes to
decline in ?meat gravy? at a pH of 6.0 and an Aw of 0.970. (Pathogen Modeling Program,
2001.) LCL- lower confidence limit. UCL- upper confidence limit.
88
Figure 10.   Acid exposure decline curve of strain 201 grown in TSB either with or without glucose or glutamine as a carbon source
and then exposed to TSB adjusted to pH 2.5 using HCl for 0-6 hr. (++, glucose + glutamine; +-, glucose only; -+ glutamine only; --,
absence of glucose + glutamine).    
89
Figure 11.   Acid exposure decline curve of strain 201 grown in TSB either with or without glucose or glutamine as a carbon source
and then exposed to TSB adjusted to pH 3.0 using HCl for 0-6 hr. (++, glucose + glutamine; +-, glucose only; -+ glutamine only; --,
absence of glucose + glutamine).    
90
Figure 12. Thermal Decline Curve of strain #204 grown either in the presence or absence of glucose (Glu) when placed in the heating
menstruum  at pH 3.0, Aw 0.987, 58?C.
91
Figure 13.  Thermal Decline Curve of strain #205 grown either in the presence or absence of glucose (Glu) when placed in the heating
menstruum at pH 3.0, Aw 0.987, 58?C.
92
Figure 14. Comparison of the 4D Values of the strains in the heating menstruum pH 3.0, Aw 0.987, 58?C.
93
Figure 15. Comparison of the 4D Values of the strains in the heating menstruum pH 7.0, Aw 0.960, 58?C.
94
Figure 16. Comparison of the T4D values of strain 201 grown in the presence of glucose and then exposed to 58?C in BHI in various
combinations of pH and Aw heating menstrua.
95
Figure 17. Comparison of the T4D values of strain 201 grown in the absence of glucose and then exposed to 58?C in BHI in various
combinations of pH and Aw heating menstrua.
96
Figure 18. Comparison of the T4D values of strain #201 grown in the presence of glucose and then exposed to 58?C in  MVJ in various
combinations of pH and Aw heating menstrua.
97
Figure 19. Comparison of the T4D values of strain #201 grown in the absence of glucose and then exposed to 58?C in  MVJ in various
combinations of pH and Aw heating menstrua.
98
Figure 20. Comparison of the T4D values of strain 201 grown in the presence of glucose (+Glu) and added to various heating menstrua
[pH (3.0 - 7.0) and Aw (0.960)] then exposed to 58?C in BHI with and without glucose (-Glu).
99
IX. Appendix
Appendix A
Identification by Listeria API Test
strains DIMa ESC "MA
N
DAR
L
XYL RHA MD
G
RIB G1P TAG
#200 - + + - - + + - - -
#201 - + + + - + + - - -
#202 - + - + - + + - - -
#203 - + + - - + + - + -
#204 - + + + - + + - - -
#205 - + + - - + + - + -
#206 - + + + - - + - - -
#207 - + + - - + + - + -
#208 - + + - - + + - + -
#209 - + + + - + + - - -
#210 - + + + - + + - - -
#211 - + + + - + + - - -
#212 - + + - - + + - - -
a DIM, Differentiation of L. Innocue and L. monocytogenes; ESC, esculin
hydrolysis; "MAN, "-mannosidase; DARL, D-arabitol; XYL, D-xylose; RHA, L-
rhamnose; MDG, methyl-"D-glucopyranoside; RIB, D-ribose; G1P, glucose-1-phosphate;
TAG, D-tagatose.
100
Appendix B  
API numerical profiles of the thirteen Listeria monocytogenes strain utilized.
Strain # Profile API Profile ID
200 6410 Listeria monocytogenes
201 6510 Listeria monocytogenes
202 2510 Listeria monocytogenes
203 6450 Listeria monocytogenes
204 6510 Listeria monocytogenes
205 6450 Listeria monocytogenes
206 6110 Listeria monocytogenes
207 6450 Listeria monocytogenes
208 6450 Listeria monocytogenes
209 6510 Listeria monocytogenes
210 6510 Listeria monocytogenes
211 6510 Listeria monocytogenes
212 6410 Listeria monocytogenes
101
Appendix C
Photographs of the API Listeria test results of ATCC strains showing positive and
negative reactionsa.
aAdapted from photographs provided by bioM?rieux.
102
Appendix D
Standard plate counts of strain #201 grown in TSB with either glucose or glutamate as a
carbon source with subsequent exposure to TSB adjusted to pH 2.5 using HCl.
Sample (gluc & glut)a  (gluc only)  (glut only) (
neither
)
Hr BHIA MVJ BHIA MVJ BHIA MVJ BHIA MVJ
0 7.10 7.12 7.16 7.17 5.97 5.85 6.15 5.50
2 6.98 6.89 7.09 6.90 2.51 2.08 2.93 2.43
4 6.84 6.41 6.93 6.51 <1.00 <1.00 1.78 <1.00
6 6.69 4.54 6.65 4.46 <1.00 <1.00 <1.00 <1.00
a. All sample were plated on both brain heart infusion agar (BHIA) and modified
vogel johnson agar (MVJ)
103
Appendix E
Standard plate counts of strain #201 grown in TSB with either glucose or glutamate as a
carbon source with subsequent exposure to TSB adjusted to pH 3.0 using HCl .
Sample (gluc & glut)a  (gluc only)  (glut only) (
neither
)
Hr BHIA MVJ BHIA MVJ BHIA MVJ BHIA MVJ
0 7.13 7.19 7.12 7.14 6.03 5.82 5.86 5.71
2 7.10 6.94 7.10 6.94 4.29 4.06 4.37 4.07
4 6.87 6.67 6.92 6.76 3.00 2.62 3.15 2.89
6 6.92 5.13 6.93 5.04 2.00 <1.00 2.28 1.60
a. All sample were plated on both brain heart infusion agar (BHIA) and modified
vogel johnson agar (MVJ)
104
Appendix F
Thermal decline of strain #201 cells in logarithmic growth phase at 58?C followed by
exposure to a heating menstruum of pH 3.0, Aw 0.960.
Sample BHIA
Time (sec) Log [cfu/ml]
0 6.42
16 5.15
32 4.60
48 4.25
64 3.88
80 3.85
96 3.54
112 3.51
128 3.08
144 3.08
160 2.85
176 2.48
192 2.30
105
Appendix G
Preliminary Study #4 growth of strain #201 and pH decline, Set A - early growth period.
Rep A Rep B Rep C
 + gluc w/o gluc  + gluc w/o gluc  + gluc w/o gluc
Hour pH ct pH ct pH ct pH ct pH ct pH ct
0 7.14 5.42 7.28 5.17 7.25 5.17 7.35 5.36 7.32 5.38 7.40 5.25
2 7.09 5.97 7.11 5.77 7.09 6.01 7.14 5.93 7.10 5.92 7.17 5.96
4 7.08 6.76 7.15 6.78 7.11 6.84 7.16 6.77 7.12 6.84 7.15 6.80
6 6.55 8.11 7.07 7.87 6.57 8.14 7.10 8.00 6.57 8.09 7.10 8.01
8 6.36 8.78 6.97 8.47 6.32 8.82 6.97 8.52 6.32 8.85 6.97 8.51
9.5 5.38 8.91 6.95 8.50 5.38 8.86 6.96 8.55 5.45 8.91 6.97 8.55
23.5 4.11 9.36 6.99 8.54 4.12 9.25 7.00 8.92 4.18 9.37 7.01 8.66
Preliminary Study #4 growth of strain #201 and pH decline, Set B - late growth period.
Rep D Rep E Rep F
 + gluc w/o gluc  + gluc w/o gluc  + gluc w/o gluc
Hour pH ct pH ct pH ct pH ct pH ct pH ct
0 7.10 6.34 7.19 5.55 7.16 5.42 7.25 5.40 7.32 5.39 7.25 5.31
14 4.43 9.15 6.98 8.74 4.41 8.98 6.98 8.69 4.45 9.20 6.96 8.67
15 4.39 9.10 6.91 8.71 4.38 9.15 6.91 8.71 4.33 9.31 6.90 8.73
16 4.35 9.40 6.92 8.64 4.33 9.32 6.92 8.76 4.33 9.38 6.94 8.68
17 4.27 9.08 6.93 8.70 4.28 9.32 6.94 8.81 4.28 9.27 6.94 8.81
18 4.23 9.35 6.94 8.65 4.25 9.31 6.93 8.67 4.24 9.26 6.94 8.92
20 4.22 9.47 6.93 8.79 4.21 9.18 6.93 8.75 4.20 9.60 6.94 8.61
22 4.17 9.32 6.92 9.06 4.18 9.40 6.94 9.01 4.18 9.52 6.99 8.92
23.7 4.15 9.45 6.95 9.02 4.14 9.35 6.97 8.85 4.13 9.43 6.95 9.39
106
X. Glossary of Terms
D Value- time required for a 1 log reduction of a microorganism at a given temperature.
T4D- The heat process at a given temperature to reduce the population by 10-4, 
equivalent to 4 times the D value + the lag time if present. 
Tlag- the duration of the shoulder of the thermal inactivation curve
?Hurdle? Technology- manipulation of multiple environmental factors to inhibit 
microbial growth
Intrinsic Factors of Foods-pH, water activity, and nutrients
Extrinsic Factor of Foods-temperature, presence of other bacteria
Aw- Water Activity is the fraction of the vapor pressure of the free water in a food over 
the vapor pressure of pure water. The water activity represents the amount of 
free water available in a food for potential use by a microorganism.
HACCP- Hazard Analysis and Critical Control Point System
107
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