The effect of sublethal exposure to peroxyacetic acid (PAA) sanitizer on adaptation to peroxidative stress and development of thermal cross-resistance was investigated in Escherichia coli O157:H7. Acute sublethal PAA sanitizer exposure was used to represent a contact scenario. Cultures were grown in Trypticase soy-yeast extract broth. Acute treatment cultures were pretreated with 0.1% PAA, then all cultures were challenged at either 80 mM H 2 O 2 or 54°C. Acute and peroxide control cultures showed substantially increased peroxidative tolerance ( D 80mM > 2 h) versus negative control cultures not exposed to sanitizer ( D 80mM = 0.19 ± 0.03 h). The inactivation rate of the acetic acid control ( D 80mM = 0.21 ± 0.05 h) was similar to the negative control rate. Acute ( D 54°C = 0.55 ± 0.07 h) cultures did not exhibit increased thermal resistance versus the control ( D 54°C = 0.54 ± 0.07 h). Thermal injury was determined as difference in D 54°C value (Δ D 54°C ) obtained on pyruvate and deoxycholate media. Thermal-induced injury was not observed in either control (Δ D 54°C = 0.04 h) or acute (Δ D 54°C = 0.05 h) cultures.

A visual DNA probe for the detection of enterotoxigenic E. coli (LT EEC) by colony hybridization was evaluated to determine its efficacy as a more restrictive, routine indicator method for foods and wastewater. The E. coli heat labile enterotoxin (LT) gene was used as the DNA probe to detect LT EEC. In control experiments the reliability of the probe was demonstrated with food spiked with LT EEC. Raw oysters and wastewater examined for naturally occurring LT EEC showed significant levels of probe positive isolates. Despite some problems, for example background noise associated with food type, the DNA probe proved satisfactory as a method for indicating the presence of enterotoxigenic Enterobacteriaceae .

The association of [ 32 P] with Clostridium botulinum 52A vegetative cells following growth in a medium containing either sodium dihydrogen [ 32 P]-pyrophosphate ([ 32 P]-SAPP) or sodium dihydrogen [ 32 P]-orthophosphate ([ 32 P]-orthophosphate) was studied. Absorbency measurements at 630 nm were used in addition to [ 32 P] recovery in determining [ 32 P] association with cellular growth and metabolism. Radiolabeling experiments showed [ 32 P]-orthophosphate was associated with vegetative cells during logarithmic growth, yet was released once stationary phase was attained or upon lysis. [ 3P ]-SAPP was also associated with cells during growth, but was not released once stationary phase was attained. Results suggested [ 32 P]-SAPP continued to bind cells or other metabolic materials following attainment of the stationary phase of cells. Fractionation of 24 and 48 h-old cultures grown in the presence of [ 32 P]-SAPP showed a higher percentage of [ 32 P] associated with the RNA fraction (3.91 and 2.48%, respectively) compared to the DNA fraction (0.09 and 0.07%, respectively).

Mesophilic aerobic microbial populations in fresh ground beef were enumerated with a new system, Petrifilm™ SM Plates (PSM), and with the conventional aerobic plate count (APC) method using standard methods agar (SMA). Total colony-forming units were determined in 119 fresh ground beef samples (29 extra-lean, 30 lean and 60 regular) purchased at nine different retail markets over a period of 6 wk. Linear regression analysis of PSM vs. APC counts gave a slope of 0.963, an intercept of −0.027, and a correlation coefficient of 0.951. Mean log 10 counts on PSM were 5.86 compared to 6.11 on SMA (P<0.01) or a mean log 10 difference of −0.25. These analyses indicate that the Petrifilm SM method would be a possible alternative for the aerobic plate count method.

The influence of 0.4% sodium acid pyrophosphate (SAPP) or 0.26% potassium sorbate (PS) on Clostridium botulinum 52A growth and toxicity from spores was studied at two pH levels 5.55 and 5.85. Absorbancy measurements at 630 nm were used in combination with microscopic evaluations and toxin analysis to compare effects of additives on normal cell development. Treatment cultures containing 0.4% SAPP and 0.26% PS at a higher pH of 5.85 showed no increase in absorbancy and no sign of toxicity, but elongated vegetative cells (≥9 μm) were observed using phase contrast microscopy rather than scanning electron microscopy. The SAPP–PS treatment culture at a lower pH of 5.55 displayed no signs of growth spectrophometrically or microscopically, as well as no toxicity. These data suggest that a SAPP–PS combination in a laboratory medium at pH 5.85 does not halt germination and outgrowth, yet may prevent cell division; whereas, the same treatment at pH 5.55 inhibits normal spore germination.

Growth response from spores and vegetative cells of Clostridium botulinum strain 52A in peptone-yeast extract-glucose (PYEG) broth at two pH levels (5.55 or 5.85) containing sodium acid pyrophosphate (SAPP) (0, 0.2, 0.4%), NaCl (0, 1.25, 2.50%) and/or potassium sorbate (KS) (0, 0.13, 0.26%) was measured as the mean A 630 nm of 20 tubes at 37°C. Additional treatments contained KC1 and MgCl 2 (0, 1.25, 2.50%) without SAPP or KS. Growth ratios (GR = treatment/control) based on time to reach A 630 = 0.35 were calculated to compare effects of additives on strain 52A. Growth from spores was affected significantly (p≤0.01) by pH level. KS and KS/pH interactions were also significant factors in growth from both spores and vegetative cells; SAPP/pH interactions were significant for cell growth, only. Combinations of SAPP (0.2, 0.4%) NaCl (0%) and KS (0.26%) were the most favorable treatments for delaying growth from spores or vegetative cells. NaCl (1.25, 2.50%) decreased antibotulinal effects produced by combinations of SAPP and KS. Elimination of NaCl enhanced antibotulinal effects. Formulations containing KC1 or MgCl 2 (without SAPP and KS) at the same molarity as the NaCl in earlier treatments (0.21, 0.43) resulted in inhibition of growth from vegetative cells greater than growth from spores in the presence of MgCl 2 at M = 0.43 (ionic strength = 1.29). This inhibition was more evident at pH 5.55 than pH 5.85. This study in a model system suggests ionic strength and/or chloride salt may be important considerations when manipulating formulations of additives designed to control C. botulinum growth.

A new system, Petrifilm VRB, was compared to two commonly used methods for enumerating coliforms: (a) colony counts on violet red bile agar (VRBA) and (b) MPN determinations with lauryl sulfate tryptose broth (LST) and brilliant green lactose bile broth (BGLB). Petrifilm VRB consists of a bottom film coated with VRB nutrients and a top film coated with a cold-water-soluble gelling agent and a tetrazolium indicator dye. Gas bubbles from lactose fermentation produced around colonies differentiated coliforms from other gram-negative organisms. Coliforms from 120 raw milk samples were enumerated by the Petrifilm VRB, VRBA and MPN methodologies. Randomly selected colonies were chosen from each methodology for characterization. Counts determined with Petrifilm VRB were comparable to counts with VRBA, and both Petrifilm VRB and VRBA compared similarly to the MPN method. Data examined by plotting log 10 coliform counts from Petrifilm VRB vs. log 10 coliform counts from VRBA resulted in a regression line with a slope of 0.959 and an intercept of −0.076 which was close to the line of equality (slope of one and intercept of zero) for the two methods. No method selected or excluded any one type of microorganisms, and VRBA and Petrifilm VRB had equivalent repeatability.

Germination of spores of Clostridium botulinum strains 62A, 213B and 12885A was monitored in modified peptone yeast extract broth flushed with CO 2 , N 2 , CO 2 + H 2 and N 2 + H 2 . Carbon dioxide enhanced germination of spores of each of the strains. Hydrogen gas in combination with CO 2 or N 2 did not substantially alter germination compared to germination in CO 2 or N 2 alone, even though the oxidation-reduction potential of the system was lower in the systems flushed with H 2 .

Sodium nitrite, sorbic acid, potassium sorbate and polyphosphates (sodium acid pyrophosphate, SAPP; sodium hexametaphosphate, SHMP; and sodium tripolyphosphate, STPP) were tested at similar preadjusted (before cooking) pH levels (in the range of pH 5.78 to 6.19 after cooking) to determine effective combinations capable of controlling Clostridium botulinum growth and toxin production in mechanically deboned chicken meat frankfurter emulsions incubated at 27°C. In combination with low levels of nitrite (40 ppm), potassium sorbate (0.26%, pH 6.06) was more effective than sorbic acid (0.20%, pH 6.03) in delaying toxin production (>27 d vs. 6 d) and in controlling growth. In formulations containing combinations of nitrite (40 ppm) and sorbic acid (0.20%) or nitrite (40 ppm) and potassium sorbate (0.26%), the addition of polyphosphates (0.4%) resulted in a greater delay of toxin production (8 to 25 d for nitrite-sorbic acid-SAPP vs. 28 d for nitrite-potassium sorbate-SAPP) at similar pH levels. Under these conditions, SAPP delayed production of detectable toxin longer (25 d) than did either SHMP (6 to 11 d) or STPP (4 to 14 d). The addition of polyphosphates to nitrite-free emulsions containing sorbic acid (0.20%) or potassium sorbate (0.26%) did not delay the development of botulinal toxin when the pH was essentially equivalent in the range of 5.78 to 6.07.

Germination of spores of Desulfotomaculum nigrificans was studied by measuring reduction in numbers of heat-resistant units. Complete (>99.9%) germination was observed with heat-activated spores suspended in a combination of 1% soytone, 0.1% ferric citrate, 0.1% sodium metabisulfite (Na 2 S 2 O 5 ), and distilled water. In this medium spores germinated most rapidly at pH 6.0 – 8.0 when incubated at 55°C after the spores were exposed to a 15 – 20 min heat-shock at 100°C. Twelve amino acids triggered germination either together or individually only in the presence of ferric citrate and Na 2 S 2 O 5 . No one amino acid as a germinant was superior to the others evaluated. Of nine carbohydrates examined (at 1% levels), fructose, ribose, and arabinose initiated germination individually in distilled water. Ferrous ion initiated germination whereas the ferric ion did not. Cu ++ (10mM) initiated germation whereas Zn ++ (10mM) inhibited germination. Phosphate buffer (67mM) and EDTA (10mM) inhibited cation-initiated germination. Reducing agents such as Na 2 S 2 O 5 may provide ferrous ions needed for spore germination. Ferrous ions as germinants indicate a possible significant role for an iron source when enumerating D. nigrificans spores or when evaluating food spoilage caused by D. nigrificans .

Injury has long been recognized in bacterial spores, especially in evaluation of apparent survival after administration of treatments to control these resistant entities. Compared to vegetative cells, the complexity of the germination and outgrowth processes has retarded research activity on injury and resuscitation. Heat-injury has been observed and studied to the greatest extent, but irradiation and chemical treatments also damage spores from anaerobic or aerobic bacteria. Injury has been associated with germination or specific steps in outgrowth or both. Damage of enzymes, DNA, RNA, membranes or other systems may be implied by resuscitation studies. Injury has been manifested by increased sensitivity to selective or antimicrobial agents or by increased requirements for germination and growth. The need for extensive fundamental research on bacterial spore injury continues to exist, especially to aid in explaining unique spore resistance.

During the last 30 years sorbate has been tested and used widely in the preservation of various food products throughout the world. Currently it has received increased attention as a potential replacer of nitrite for botulism control in processed meat products. Previous reports, however, had suggested sorbate as a selective agent for clostridia in laboratory media. Recent developments as well as the need for safe, practical and effective food preservatives in current and future food processing have generated intense interest in preservatives such as sorbate. This paper reviews the significant developments relating to use of sorbate as a food preservative - its antimicrobial effects, applications, advantages and limitations. A summary of the current status as well as unanswered questions relevant to the mechanism(s) through which the compound exerts its antimicrobial activity also is presented.

Growth and survival of Clostridium perfringens indigenous to raw beef were compared to that of a composite of eight strains of C. perfringens inoculated into raw ground beef round, beef-round strips, and beef-round cores. A constantly rising temperature rate of 6 C/h and a dynamic rising temperature rate, predetermined from long time-low temperature (LTLT) oven beef cookery processes, were used to evaluate growth and survival. Survivors of C. perfringens were observed in raw ground beef and autoclaved ground beef at temperatures between 59 and 63 C. Growth in raw ground beef was more rapid than in raw beef-round strips. Raw ground beef supported growth of the indigenous flora to a greater extent than did autoclaved ground beef inoculated with the composite. The populations in raw, domestic frozen beef rounds used in commercial cooking processes were low, ranging from < 70/g to 1000/g, and varied among lots.

Nine strains of Clostridium perfringens were compared for phospholipase C production in autoclaved ground beef (AGB) at 37 C. Enzyme production is reported as units per colony forming unit (CFU). Phospholipase C concentrations were determined by the hemolysin indicator method and colonies were observed on Tryptose Sulfite Cycloserine agar. Enzyme production by each of the nine strains was significantly different. The increase and decrease of viable cells of C. perfringens in AGB exposed to temperatures rising constantly at a rate of 4.1 C/h was accompanied by the appearance and disappearance of phospholipase C. This demonstrated that C. perfringens can produce detectable levels of phospholipase C when grown during dynamic increasing temperatures.

Nitrite plays a major role in the botulinal safety of cured meat products. When used at appropriate levels, it retards Clostridium botulinum growth and delays production of its deadly neurotoxin. Even though the incidence of botulinal spores in meat is very low, factors such as the tonnage of cured meats consumed, the potential for mishandling such products, and the nature of the disease necessitate the use of nitrite or other equally effective compound(s) for extra safety. Residual nitrite and nitrosamine levels in cured meat products have been decreasing in recent years through control and research conducted by the meat industry and related institutions. Such levels are minimal compared to total nitrate and nitrite amounts ingested or formed in the human body. Sorbate, especially in combination with nitrite at concentrations adequate only for cured meat color and flavor development, is at least as effective as currently used nitrite levels in delaying C. botulinum growth and toxin production. The mechanism(s) through which nitrite and/or sorbate perform their actions upon C. botulinum remains to be found. A review of the pertinent research completed to date should be helpful in the search for the mechanism(s).

New social, economic and political demands for conservation of energy, water and consumable products coupled with changes in lifestyle including more meals eaten away from home will require new approaches to food handling. These modifications may increase or uncover new hazards and potential opportunities for food borne illness. Microbial hazards will remain a major problem but will be only one of the many concerns of the consumer about food. Increases in awareness of newly identified pathogens, carcinogens, mutagens and the like will demand greater efforts but will also increase cost of foods. Acute sensitivity to escalating costs will in turn bring about more objective evaluations of benefit/risk ratios on all programs. Education of producers, handlers, processors and consumers will be required so that they may monitor and serve as protectors of the food system thus minimizing regulatory costs and placing responsibility at the point of action. This process will be successful only with appropriate educational and research support to evaluate and implement modified programs.

Inactivation of a composite of five serotypes of salmonellae was monitored in autoclaved ground beef exposed to constantly rising temperatures increased at rates similar to those used in beef cookery. Rising temperature rates of 6.0 C/h, 8.5 C/h and 12.5 C/h and constant temperatures of 55, 57, 61 and 63 C were examined. Survival of Salmonella typhimurium TM-1 was compared to survival of the composite. D and z values were compared for constant and rising temperature rates. The D 50 C for constant temperature data was 30.2 min, and the D 50 C for changing temperature data was 78.6 min (6.0 C/h), 82.4 min (8.5 C/h), and 49.8 min (12.5 C/h). Neither serotype nor heat treatment of ground beef had a major influence on apparent heat resistance of salmonellae. A comparison of these results to previous rising temperature work with Clostridium perfringens suggested that controlling C. perfringens will result in control of salmonellae. On the basis of these results, the July 18, 1978, USDA processing ruling appears adequate to control salmonellae in precooked beef roasts.

Growth of an eight-strain composite of Clostridium perfringens was compared using rolled raw beef strips, raw and autoclaved ground beef and Fluid Thioglycollate medium. The inoculated media were exposed to either static or dynamic temperatures increasing at linear rates. The most rapid growth and shortest lag times were observed in autoclaved ground beef. Generation times in the beef media were directly related to oxidation-reduction potential. Fluid Thioglycollate medium supported the slowest growth even though it presented a favorable E h .

Alkaline-earth type films, produced by detergent-water interactions, are frequently deposited on eating utensils during mechanical dishwashing. These films are aesthetically unacceptable but their public health significance and food spoilage potential have not been established. Sterile glass petri dishes were washed in an institutional-type dishmachine containing Bacillus subtilis spores in the washwater. A film-producing model system was developed to examine film formation in conjunction with spore deposition. The influence of three distinct detergent formulations on this association was also determined. Detergent formulations contained 6.75% phosphorus (P), as sodium tripolyphosphate (STP), 3.0% P as STP, a proprietary phosphate substitute, and the film-producing model formulation devoid of both STP and substitute water conditioning agents. Film deposition was quantified as μ g Ca ++ per cm 2 by an acid rinse-atomic absorption spectrophotometric method. The quantity of B. subtilis spores recovered from washed petri dishes in the model system was related to film deposition. The relationship was dependent on the number of consecutive dishmachine cycles and the hardness of the water supply. Above a threshold value of 3 μ g Ca ++ per cm 2 , Ca ++ deposition and B. subtilis spore harborage were directly correlated. Both of these conditions were inhibited to varying degrees by the three detergent formulations. These data suggest that dishware filming may be cause for public health and food spoilage concerns.

Recognition of sublethally impaired microorganisms is essential to practical interpretations of data in food microbiology. Sublethal injury induced by exposure to environmental stress often is demonstrated as a loss by the microorganism of one or more abilities to function characteristically under conditions that are satisfactory for untreated cells. Various treatments related to food processing such as heat; cold; freezing-thawing; freeze-drying; moisture reduction; irradiation; and exposure to food environments, sanitizers. or preservatives may induce sublethal damage in cells or spores. Many cellular modifications may be observed in injured cells. Included are susceptibility to selective agents and antimicrobials, leakage of intracellular material, and modified metabolic activities. Injury is characterized by the capability of the microorganism to return to normalcy during a resuscitation process in which the damaged essential components are repaired. Sublethal damage in spores must be considered specially because of the complexity of the spore entity and its characteristic high resistance to stress. Extent of sublethal damage and mechanisms of injury and repair are varied and related to conditions of stress and resuscitation. Knowledge of sublethal injury is indispensable in evaluating laboratory data, in developing or modifying food processes, and in preservating culture activity.