Growth of Listeria monocytogenes in Thawed Frozen Foods.

The growth characteristics of Listeria monocytogenes inoculated onto frozen foods (corn, green peas, crabmeat, and shrimp) and thawed by being stored at 4, 8, 12, and 20°C were investigated. The growth parameters, lag-phase duration (LPD) and exponential growth rate (EGR), were determined by using a two-phase linear growth model as a primary model and a square root model for EGR and a quadratic model for LPD as secondary models, based on the growth data. The EGR model predictions were compared with growth rates obtained from the USDA Pathogen Modeling Program, calculated with similar pH, salt percentage, and NaNO2 parameters, at all storage temperatures. The results showed that L. monocytogenes grew well in all food types, with the growth rate increasing with storage temperature. Predicted EGRs for all food types demonstrated the significance of storage temperature and similar growth rates among four food types. The predicted EGRs showed slightly slower rate compared with the values from the U.S. Department of Agriculture Pathogen Modeling Program. LPD could not be accurately predicted, possibly because there were not enough sampling points. These data established by using real food samples demonstrated that L. monocytogenes can initiate growth without a prolonged lag phase even at refrigeration temperature (4°C), and the predictive models derived from this study can be useful for developing proper handling guidelines for thawed frozen foods during production and storage.

Evaluation of an enzyme-linked immunosorbent assay (ELISA) kit for the detection of botulinum neurotoxins A, B, E, and F in selected food matrices.

The mouse bioassay (MBA) is the only accepted standard method for detection of botulinum neurotoxins (BoNTs) in foods. The ELISA method has several advantages over the MBA and is therefore widely used for in vitro detection of BoNTs. The US Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) conducted a precollaborative study to evaluate the applicability of Botulinum Toxin ELISA kits for the detection of BoNT serotypes A, B, E, and F in a variety of food matrices. In this study, food samples (e.g., broccoli, salami, smoked salmon, green beans, orange juice, tomato juice, low-fat plain yogurt, whole milk, liquid infant formula milk, and liquid eggs) were spiked with high, medium, and low concentration BoNT serotypes A, B, E, and F. Samples (unspiked and spiked) were tested at both laboratories by the ELISA kits. All food samples were positive for BoNTs by ELISA in both laboratories at medium and high spiking levels; a positive ELISA result in low spiked samples was both serotype and laboratory dependent. Overall, the ELISA method appears to be an effective preliminary screening method for BoNT detection in food matrices.

Issues to consider when setting intervention targets with limited data for low-moisture food commodities: a peanut case study.

Peanuts and peanut-containing products have been linked to at least seven salmonellosis outbreaks worldwide in the past two decades. In response, the Technical Committee on Food Microbiology of the North American Branch of the International Life Sciences Institute collaborated with the American Peanut Council to convene a workshop to develop a framework for managing risk in low-moisture food commodities where large data sets are unavailable (using peanuts as the example). Workshop attendees were charged with answering questions regarding the appropriate statistical and scientific methods for setting log reduction targets with limited pathogen prevalence and concentration data, suitable quantities of data needed for determining appropriate log reduction targets, whether the requirement of a 5-log reduction in the absence of data to establish a target log reduction is appropriate, and what targeted log reduction would protect public health. This report concludes that the judgment about sufficient data is not solely scientific, but is instead a science-informed policy decision that must weigh additional societal issues. The participants noted that modeling efforts should proceed with sampling efforts, allowing one to compare various assumptions about prevalence and concentration and how they are combined. The discussions made clear that data and risk models developed for other low-moisture foods like almonds and pistachios may be applicable to peanuts. Workshop participants were comfortable with the use of a 5-log reduction for controlling risk in products like peanuts when the level of contamination of the raw ingredients is low (<1 CFU/g) and the process well controlled, even when limited data are available. The relevant stakeholders from the food safety community may eventually conclude that as additional data, assumptions, and models are developed, alternatives to a 5-log reduction might also result in the desired level of protection for peanuts and peanut products.

Variation in Listeria monocytogenes dose responses in relation to subtypes encoding a full-length or truncated internalin A.

Internalin A (InlA; encoded by inlA) facilitates the crossing of the intestinal barrier by Listeria monocytogenes. Mutations leading to a premature stop codon (PMSC) in inlA and thus attenuated mammalian virulence have been reported. We recently characterized 502 L. monocytogenes food isolates from a retail survey and 507 human clinical isolates from multiple U.S. states with respect to the presence/absence of inlA mutations. The objective of this study was to investigate the hypothesis that dose responses for human listeriosis vary between L. monocytogenes strains with and those without a PMSC in inlA. Subtype-specific prevalence and concentration distributions in food, along with epidemiologic and consumption data, were input into established dose-response models to generate an r value (probability of a cell causing illness). Under the conservative assumption that L. monocytogenes levels at retail represent levels consumed, mean log(10) r values were -8.1 and -10.7 for L. monocytogenes subtypes with genes encoding a full-length and a truncated InlA, respectively. L. monocytogenes carrying a 5' frameshift mutation in a homopolymeric tract showed a mean log(10) r value of -12.1. Confidence intervals for the r values and their differences varied depending on subtypes. When the increase in concentration of L. monocytogenes subtypes between retail and consumption was considered, mean log(10) r values were reduced to -10.4, -13.8, and -12.8 for the subtypes with genes encoding a full-length InlA, for the subtypes carrying a PMSC in inlA, and for all L. monocytogenes isolates regardless of subtype, respectively. Our study provides further quantitative evidence that L. monocytogenes subtypes vary in abilities and relative likelihoods of causing human disease, which were mechanistically related to defined genetic markers.

The Key Events Dose-Response Framework: its potential for application to foodborne pathogenic microorganisms.

The Key Events Dose-Response Framework (KEDRF) is an analytical approach that facilitates the use of currently available data to gain insight regarding dose-response relationships. The use of the KEDRF also helps identify critical knowledge gaps that once filled, will reduce reliance on assumptions. The present study considers how the KEDRF might be applied to pathogenic microorganisms, using fetal listeriosis resulting from maternal ingestion of food contaminated with L. monocytogenes as an initial example. Major biological events along the pathway between food ingestion and the endpoint of concern are systematically considered with regard to dose (i.e., number of organisms), pathogen factors (e.g., virulence), and protective host mechanisms (e.g., immune response or other homeostatic mechanisms). It is concluded that the KEDRF provides a useful structure for systematically evaluating the complex array of host and pathogen factors that influence the dose-response relationship. In particular, the KEDRF supports efforts to specify and quantify the sources of variability, a prerequisite to strengthening the scientific basis for food safety decision making.

Development and validation of a predictive model for Listeria monocytogenes Scott A as a function of temperature, pH, and commercial mixture of potassium lactate and sodium diacetate.

The objective of this study was to develop and validate secondary models that can predict growth parameters of L. monocytogenes Scott A as a function of concentrations (0-3%) of a commercial potassium lactate (PL) and sodium diacetate (SDA) mixture, pH (5.5-7.0), and temperature (4-37oC). A total of 120 growth curves were fitted to the Baranyi primary model that directly estimates lag time (LT) and specific growth rate (SGR). The effects of the variables on L. monocytogenes Scott A growth kinetics were modeled by response surface analysis using quadratic and cubic polynomial models of the natural logarithm transformation of both LT and SGR. Model performance was evaluated with dependent data and independent data using the prediction bias (Bf) and accuracy factors (Af) as well as the acceptable prediction zone method [percentage of relative errors (%RE)]. Comparison of predicted versus observed values of SGR indicated that the cubic model fits better than the quadratic model, particularly at 4 and 10oC. The Bf and Af for independent SGR were 1.00 and 1.08 for the cubic model and 1.08 and 1.16 for the quadratic model, respectively. For cubic and quadratic models, the %REs for the independent SGR data were 92.6 and 85.7, respectively. Both quadratic and cubic polynomial models for SGR and LT provided acceptable predictions of L. monocytogenes Scott A growth in the matrix of conditions described in the present study. Model performance can be more accurately evaluated with Bf and Af and % RE together.

Modeling the physiological state of the inoculum and CO2 atmosphere on the lag phase and growth rate of Listeria monocytogenes.

In previous studies, the growth of L. monocytogenes has been modeled under different CO2 headspace concentrations; however, the inoculum cells were always in the stationary phase. In this study, the growth of L. monocytogenes under different CO2 concentrations as affected by the physiological state of the cells was investigated. Exponential-growth-phase, stationary-phase, dried, and starved cells were prepared and inoculated at 5 degrees C into brain heart infusion broths that had been preequilibrated under atmospheres of 0, 20, 40, 60, or 80% CO2 (the balance was N2). Lag-phase duration times (LDTs) and exponential growth rates were determined by enumerating cells at appropriate time intervals and by fitting the data to a three-phase linear function that has a lag period before the initiation of exponential growth. Longer LDTs were observed as the CO2 concentration increased, with no growth observed at 80% CO2. For example, the LDTs for exponential-phase, stationary-phase, starved, and dried cells were 2.21, 8.27, 9.17, and 9.67 days, respectively, under the 40% CO2 atmosphere. In general, exponential-growth-phase cells had the shortest LDT followed by starved cells and stationary-phase cells. Dried cells had the longest LDT. Exponential growth rates decreased as the CO2 concentrations increased. Once exponential growth was attained, no retained differences among the various initial physiological states of the cells for any of the atmospheres were observed in the exponential growth rates. The exponential growth rates under 0, 20, 40, 60, and 80% CO2 averaged 0.39, 0.37, 0.23, 0.23, and 0.0 log CFU/day, respectively. Dimensionless factors were calculated that describe the inhibitory action of CO2 on the LDTs and exponential growth rates for the various physiological states.

Attributing risk to Listeria monocytogenes subgroups: dose response in relation to genetic lineages.

The objective of this study was to evaluate the hypothesis that the dose-response relationship for Listeria monocytogenes in humans varies with genotypic lineage or subtype. The linkages between molecular subtyping data and enumeration data for L. monocytogenes subtypes in foods consumed by the at-risk population were examined to test this hypothesis. We applied a conditional probability model to conduct a subtype-specific dose-response analysis, with the focus on invasive listeriosis. L. monocytogenes differed not only in the molecular subtype and lineage but also in the contamination level when isolates of the pathogen occurred in retail samples of ready-to-eat foods. Using the exponential model parameter r-value as a measure (essentially the probability of a single cell causing illness), we found that the virulence varied among L. monocytogenes lineages by several orders of magnitude. Under the assumptions made, for L. monocytogenes lineages I and II the consumption of a single cell would result in listeriosis with log average probabilities of -7.88 (equivalent to once in 10(7.78) times) and -10.3, respectively, as compared with -9.72 for L. monocytogenes independent of subtype. A greater difference in r-values was found for selected ribotypes. The uncertainty about the r-value estimates was small compared with the large differences in the virulence parameters themselves. Thus, for L. monocytogenes both subtype and the number of cells consumed matter, highlighting the usefulness of considering both exposure concentration and subtype prevalence in dose-response analysis. As advances are made in molecular subtyping and quantitative tools for dose-response analysis, further studies integrating genomic data into quantitative risk assessments will enable better attribution of disease risk to L. monocytogenes subtypes.

Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies.

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.

Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis.

The suitability and sensitivity of two in vitro lateral-flow assays for detecting Clostridium botulinum neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a C. botulinum contamination event.

Methods for detection of Clostridium botulinum toxin in foods.

Botulism is a deadly disease caused by ingestion of the preformed neurotoxin produced from the anaerobic spore-forming bacteria Clostridium botulinum. Botulinum neurotoxins are the most poisonous toxins known and have been a concern in the food industry for a long time. Therefore, rapid identification of botulinum neurotoxin using molecular and biochemical techniques is an essential component in the establishment of coordinated laboratory response systems and is the focus of current research and development. Because of the extreme toxicity of botulinum neurotoxin, some confirmatory testing with the mouse bioassay is still necessary, but rapid methods capable of screening large numbers of samples are also needed. This review is focused on the development of several detection methods for botulinum neurotoxins in foods.

Effect of prior growth conditions on the thermal inactivation of 13 strains of Listeria monocytogenes in two heating menstrua.

The thermal tolerance of 13 Listeria monocytogenes strains was tested using a submerged heating coil apparatus. The strains were grown individually for 18 h at 37 degrees C in acidogenic tryptic soy broth (without dextrose) supplemented with 1% glucose and 1% glutamine (TSB+G) or nonacidogenic tryptic soy broth supplemented with 1% glutamine but containing no glucose (dextrose) (TSB-G). The former medium results in cells induced for pH-dependent, stationary-phase acid resistance, whereas the latter medium allows L. monocytogenes to grow to high numbers in the absence of glucose, yielding cells that are not induced for pH-dependent, stationary-phase acid resistance. The average final pH values of the 18-h TSB+G and the TSB-G cultures were 4.7 and 6.7, respectively. The cells grown in the acid resistance-inducing and non-acid resistance-inducing media were then tested in two heating menstrua that consisted of brain heart infusion broth adjusted to pH 3.0 and water activity (a(w)) of 0.987 or pH 7.0 and a(w) 0.970. In 14 of the 26 menstruum-strain combinations tested, the acid resistance-induced strains were more heat resistant then the equivalent noninduced cultures. No difference in the pattern of thermal resistance in response to induction of acid resistance was apparent among the different serovars tested. The results suggest that the ability of prior induction of acid resistanceto enhance thermal resistance can vary substantially among L. monocytogenes strains.

Quantitative contamination and transfer of Escherichia coli from foods by houseflies, Musca domestica L. (Diptera: Muscidae).

The housefly, Musca domestica L. (Diptera: Muscidae), is recognized as an important factor in the dissemination of various infectious diseases such as cholera, shigellosis, and salmonellosis. They can also serve as a cross-contamination vector for other foodborne pathogens. However, the potential for bacterial transfer by houseflies has been demonstrated in a qualitative rather than quantitative manner. In this study, the numbers of bacteria a housefly can carry on its body and transfer to a clean surface after exposure to a sugar-milk aqueous solution, steak, and potato salad contaminated with a fluorescent gene Escherichia coli (8 log10 CFU/ml) were determined. In the first series of experiments to quantify bacterial numbers on the flies, about 40-60 flies were transferred into a sterile cage, exposed to the food for 30 min, the flies immobilized and the attached E. coli on each fly enumerated. Detectable E. coli (>1.7 log10 CFU/fly) were found on 43% (29/67), 53% (23/43), and 62% (32/52) of the flies in the cages with sugar/milk, steak, and potato salad, respectively. For the positive flies, the geometric mean carriage (log10 CFU/fly) was 2.93+/-1.24 for sugar-milk, 3.77+/-1.28 for steak, and 2.25+/-0.64 for the potato salad. In the second series of experiments, the transfer of bacteria by individual flies from contaminated food to the inner surface of a sterile jar per each landing was determined. E. coli transferred from the sugar-milk was 3.5+/-0.7 log10 CFU/fly-landing, 3.9+/-0.7 for steak and 2.61+/-1.16 for the potato salad. From the initial contamination levels of bacteria and the number of transferred bacteria, it can be calculated that flies contaminate clean surfaces with approximately 0.1 mg of food per landing.

Thermal inactivation, growth, and survival studies of Listeria monocytogenes strains belonging to three distinct genotypic lineages.

Twenty-one Listeria monocytogenes strains belonging to three different genotypic lineages were evaluated for differences between lineages and between individual strains with respect to thermal inactivation, growth, and survival. Three sets of heat inactivation conditions (60 degrees C, pH 6.0, and 0.5 M lactate; 55 degrees C, pH 6.0, and 0.5 M lactate; and 50 degrees C, pH 4.0, and 0.5 M lactate) were used on strains grown in modified brain heart infusion (BHI) broth with and without glucose. Two sets of growth conditions (35 degrees C, pH 6.5, and 0.1 M lactate and 5 degrees C, pH 6.5, and 0.1 M lactate) were used with modified BHI broths to determine lag phases and exponential growth rates. Two sets of conditions (28 degrees C, pH 4.0, and 1 M lactate and 28 degrees C, pH 4.5, and 0.5 M lactate) were used with modified BHI broth to determine survival times (D-values). Thermal inactivation D-values were consistently lowest for lineage III, but differences were not significant for any set of conditions tested. Some significant differences were observed between lineages with respect to some of the growth and survival conditions tested. Extensive strain-to-strain variation was observed for all parameters tested. Average coefficients of variation for the thermal inactivation, growth, and survival studies were 0.31, 0.18, and 0.26, respectively. Strain-to-strain variations were approximately equal to the uncertainties associated with the analytical procedures. The results obtained indicate a diversity among strains encountered in food processing that must be accounted for in process calculations and risk assessments.

Use of Epidemiologic and Food Survey Data To Estimate a Purposefully Conservative Dose-Response Relationship for Listeria monocytogenes Levels and Incidence of Listeriosis †.

The development of effective quantitative microbial risk-assessment models for foodborne pathogens depends on the availability of data on the consumers' exposure to a biological agent and the dose-response relationship that relates levels of the biological agent ingested with frequency of infection or disease. Information on the latter has historically been acquired from human volunteer feeding studies. However, such studies are not feasible for pathogens that either have a significant risk of being life threatening or for which morbidity is primarily associated with high-risk populations (i.e., immunocompromised persons). For these pathogens, it is proposed that purposefully conservative dose-response relationships can be estimated on the basis of combining available epidemiologic data with food-survey data for a ready-to-eat product. As an example, data on the incidence of listeriosis in Germany were combined with data on the levels of Listeria monocytogenes in smoked fish to generate a dose-response curve for this foodborne pathogen.

Risk Assessment and Predictive Microbiology.

The need and desirability of being able to perform quantitative microbial risk assessments for food processing and preparation operations has been discussed extensively for the past several years. However, there has been little application of this approach in large part due to the need to account for the changes in bacterial populations as a result of food environments and processing. The use of predictive food microbiology models has the potential for overcoming these limitations. Through integration of predictive modeling with risk assessment, it is possible to estimate how changes in unit operations are likely to effect the overall safety of a food. Hypothetical examples of how these techniques could be applied to both single-step and multiple-step food-processing and preparation operations are provided.

Ability of Bdellovibrio bacteriovorus 109J to Lyse Gram-Negative Food-Borne Pathogenic and Spoilage Bacteria.

Bdellovibrios are a group of aerobic, predatory bacteria which attack, penetrate and grow in many species of gram-negative bacteria, causing the lysis of the invaded prey organism. Bdellovibrio bacteriovorus strain 109J varied in its ability to lyse 32 bacterial strains comprising six genera of food-borne pathogens and spoilage organisms. The reduction in the levels of the prey bacteria ranged from 0.1 to 7.7 log-values after 7 h of incubation at 30°C. Escherichia coli strain 2239-69 (pathogenic serotype 026:H11) was lysed most effectively at temperatures between 30 and 37°C, however, lysis also occurred at 12 and 19°C when the incubation period was extended to 24 h. Bdellovibrio bacteriovorus was effective in reducing the level of E. coli 2239-69 at pH 5.6 to 8.6. Increasing the Bdellovibrio : E. coli ratio resulted in a more rapid E. coli reduction. This study demonstrated the potential usefulness of bdellovibrios for the biological control of pathogenic and spoilage organisms in foods.

Comparison of Organic Acid Salts for Clostridium botulinum Control in an Uncured Turkey Product.

Health concerns have led consumers toward purchasing nitrite-free, low salt meat and poultry products. Lacking these barriers to control growth of bacterial pathogens, such products carry heightened risks for botulism, especially if temperature abused. To address this threat, five organic acid salts were evaluated as potential antibotulinal agents. Ground turkey breast was formulated with 1.4% NaCl, 0.3% sodium pyrophosphate, 0-6% organic acid salts, 10% ice, and 500 spores per g of a 6-strain mixture of proteolytic Clostridium botulinum . Vacuum-packaged product (10 g) was heated in 75°C water for 20 min, cooled, and incubated for up to 18 d at 28°C. Botulinal neurotoxin was detected by mouse bioassay at 2 d in samples which lacked any of the test compounds. Samples containing 2% acid salt developed neurotoxin, which was detected at 2, 2, 4, 5, and 5 d for pyruvate, citrate, lactate, acetate, and propionate, respectively. With 6% acid salt additions, samples remained neurotoxin free until 7 d with pyruvate, 18 d with citrate, and >18 d for the remaining compounds. Monocarboxylic acid salts exhibited antibotulinal activity related to their dissociation constants (pKa) Citrate did not fit this pattern, however, suggesting a different mechanism of action. This study reveals that a variety of organic acid salts possess activity that can be used alone or possibly in combination to enhance the safety of nitrite-free turkey products.

Reduction of an Escherichia coli K12 Population by Bdellovibrio bacteriovorus Under Various In Vitro Conditions of Parasite:Host Ratio, Temperature, or pH.

Intrinsic and extrinsic parameters of food products, as well as bacterial population, were evaluated for their effects on the ability of Bdellovibrio bacteriovorus , a bacterium parasitic upon gram-negative bacteria, to reduce an Escherichia coli population. High concentrations of both parasite and host were the most effective for reducing a specified E. coli population. B. bacteriovorus was able to reduce the E. coli count by 90% (1 log) in < 1 h at ratios of 5:1, 10:1, and 30:1 (parasite:host). Temperatures between 20 and 30°C were more conducive to bdellovibrio attack than temperatures less than 20°C. E. coli populations were reduced by more than 7-log values after 7 h of incubation at 30°C with parasite:host ratios of 2:1, 5:1, and 10:1. Greater than a 5-log reduction in the E. coli population was observed at the ratio of 30:1. B. bacteriovorus reduced the E. coli population by 1 log in approximately 24 min and 20 min at pH 7.2 and 6.8, respectively. At pH values <6.8, the activity of B. bacteriovorus was diminished. These results define some of the conditions where the application of B. bacteriovorus may aid in the reduction/elimination of some gram-negative pathogens and spoilage flora that may be present in foods.

Effects and Interactions of Temperature, pH, Atmosphere, Sodium Chloride, and Sodium Nitrite on the Growth of Listeria monocytogenes.

The effects and interactions of temperature (5, 19, 28, 37°C), initial pH (6.0 and 7.5), atmosphere (aerobic and anaerobic), sodium chloride content (0.5 and 4.5%), and sodium nitrite concentration (0, 50, 100, 200, 1000 µg/ml) on the growth of Listeria monocytogenes Scott A were determined using Tryptose Phosphate Broth. Growth data were analyzed by regression analysis to generate "best-fit" Gompertz equations, which were used subsequently to calculate lag phase duration, exponential growth rate, generation time, and maximum population density values. The data indicated that the growth kinetics of L. monocytogenes was dependent on the interaction of the five variables, particularly in regard to exponential growth rates and lag phase durations. The data suggest that sodium nitrite can have significant bacteriostatic activity against L. monocytogenes and may provide cured meats with a degree of protection against this microorganism, particularly if employed in conjunction with a combination of acidic pH, vacuum packaging, high salt concentrations, and adequate refrigeration.