Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization.

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

A novel strictly anaerobic recovery and enrichment system incorporating lithium for detection of heat-injured Listeria monocytogenes in pasteurized milk containing background microflora.

Heat-injured cells of Listeria monocytogenes were recovered from heated raw milk containing noninjured Enterococcus faecium by combining a simple method for obtaining strict anaerobiosis with a novel enrichment broth, Penn State University broth (PSU broth). Strictly anaerobic conditions were rapidly achieved by adding 0.5 g of filter-sterilized cysteine per liter to PSU broth and then purging the preparation with N2 gas. Little resuscitation or growth occurred in strictly anaerobic PSU broth without lithium chloride because of overgrowth by E. faecium. The growth of E. faecium decreased dramatically with increasing LiCl concentration; LiCl concentrations of 8 and 10 g/liter were completely bacteriostatic. The mechanism of inhibition by LiCl appeared to involve competition with the divalent cations Ca2+ and Mg2+. Heat-injured L. monocytogenes consistently recovered and grew rapidly in strictly anaerobic PSU broth containing 4, 6, or 7 g of LiCl per liter. The use of strictly anaerobic PSU broth containing 7 g of LiCl per liter permitted detection of severely heat-injured L. monocytogenes in one simple recovery-enrichment step by eliminating oxygen toxicity and inhibiting the growth of background microflora, without preventing the resuscitation and subsequent growth of heat-injured L. monocytogenes. L. monocytogenes heated in raw milk at 62.8 degrees C for 10, 15, and 20 min could be consistently recovered from strictly anaerobic PSU broth enrichment cultures at 30 degrees C after 48, 96, and 144 h, respectively, and hence, use of PSU broth may result in better recovery of both injured and noninjured cells from foods than currently used U.S. Department of Agriculture and Food and Drug Administration preenrichment procedures.     

Thermal inactivation of Listeria monocytogenes within bovine milk phagocytes.

Thermal resistance of intracellular and freely suspended Listeria monocytogenes that was associated with a milkborne outbreak of listeriosis was studied by using the sealed tube and slug flow heat exchanger methods. Test temperatures for the former method were 57.8, 62.8, 66.1, and 68.9 degrees C (136, 145, 151, and 156 degrees F, respectively); whereas those for the latter method were 66.1, 68.9, 71.7, and 74.4 degrees C (151, 156, 161, and 166 degrees F, respectively). The heating menstruum was sterile, whole milk. The intracellular inoculum was generated from an in vitro phagocytosis reaction by using endotoxin-induced bovine milk phagocytes. The phagocyte population consisted of 88% neutrophils, 8% macrophages, and 4% lymphocytes. Neutrophils harbored the majority of intracellular L. monocytogenes. The mean level of infectivity in the phagocyte population was 43%, and there were 26.1 +/- 19.3 bacteria per cell (10(4) viable cells per ml of test milk). Initial bacterial counts for the freely suspended and intracellular experiments (the latter was based on a sonically disrupted sample) were 10(6) L. monocytogenes cells per ml. Heat-stressed bacteria were recovered by direct plating in parallel with recovery from an enrichment broth; both methods gave comparable results. The predicted D62.8 degrees C (145 degrees F) value for intracellular sealed tube studies was 53.8 s (ZD = 5.6 degrees C [10.0 degrees F]), indicating a safe 33.4 D margin of inactivation for vat pasteurization (62.8 degrees C for 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods.

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Behaviour of Listeria monocytogenes under combined chilling processes

The behaviour of Listeria monocytogenes under chilling processes was investigated. Growth kinetics were measured at 7 degrees C in TSBYE culture medium as a function of pH (7.2 and 6.2), pre-incubation temperatures (4 or 7 degrees C), cooling (0.05 or 0.1 degree C min-1) and freezing (0 and -5 degrees C) treatments. Growth curves generated were fitted by Gompertz and Baranyi functions. The Baranyi function gave better parameter estimation values than the Gompertz equation which over-estimated the specific growth rate values. Listeria monocytogenes grew at 7 degrees C without a lag phase, except when the sub-culture was performed at 37 degrees C, whereas the specific growth rate was affected by the chilling processes. In fact, L. monocytogenes grew slightly faster at 7 degrees C when a 4 degrees C pre-incubation treatment was applied than with a 7 degrees C pre-incubation treatment. These results suggest that to mimic the processes of contamination in industry, predictive microbiology studies with L. monocytogenes should be performed with organisms cultured at low temperatures.     

Evaluation of ALOA plating medium for its suitability to recover high pressure-injured Listeria monocytogenes from ground chicken meat

AIMS: To evaluate a chromogenic plating medium for the isolation of sublethally injured cells of Listeria monocytogenes from processed foods. METHODS AND RESULTS: The inactivation of L. monocytogenes at pressures up to 400 MPa and 12 degrees C in ground chicken meat was employed to examine the recovery of high-pressure injured cells. Before and after different repair incubation periods at 30 degrees C in a nonselective broth, samples were plated onto a selective and differential agar [Agar Listeria according to Ottaviani and Agosti (ALOA)] and in the same medium supplemented with 4% sodium chloride (ALOA-S), and incubated at 37 degrees C. Sublethally injured cells were able to grow when directly plated onto the ALOA medium, without a previous repair incubation period. However, only uninjured cells grew on the ALOA-S medium. CONCLUSIONS: Sublethally injured cells of L. monocytogenes can be quantified by subtracting counts on ALOA-S medium from counts on ALOA medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Possible applications include direct enumeration on ALOA of stressed cells of L. monocytogenes in foods with more than 100 colony forming units per gram.     

Comparative recovery of uninjured and heat-injured Listeria monocytogenes cells from bovine milk

The standard selective enrichment protocols of the Food and Drug Administration (FDA) and U.S. Department of Agriculture (USDA) were compared with an experimental nonselective broth enrichment (NSB) protocol and variations of the standard cold-enrichment (CE) protocol for the recovery of heat-injured Listeria monocytogenes. Bacterial cells (10(7)/ml) were suspended in sterile milk and heated at 71.7 degrees C in a slug-flow heat exchanger for holding times ranging from 1 to 30 s. Surviving cells were determined (50% endpoint) by the given protocols, and the following D values were obtained: NSB, D = 2.0 +/- 0.5 s; FDA, D = 1.4 +/- 0.3 s; USDA, D = 0.6 +/- 0.2 s; CE, D less than or equal to 1.2 s. The respective direct-plating media used in these enrichments were also analyzed for recovery, and the following D values were calculated from the enumeration of surviving cells; NSB, D = 2.7 +/- 0.8 s; FDA, D = 1.3 +/- 0.4 s; USDA, D = 0.7 +/- 0.2 s. The low levels of heat-injured L. monocytogenes cells which were detected at inactivation endpoints on the optimal nonselective media (25 degrees C for 7 days) failed to recover and multiply during experimental CEs (4 degrees C for 28 days). Initial inactivation experiments in which raw whole milk was used as the heating menstruum gave much lower recoveries with all protocols. The detectable limits for uninjured cells that were suspended in raw milk were similar (0.35 to 3.2 cells per ml) for the standard CE, FDA, and USDA protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative recovery of uninjured and heat-injured Listeria monocytogenes cells from bovine milk.

The standard selective enrichment protocols of the Food and Drug Administration (FDA) and U.S. Department of Agriculture (USDA) were compared with an experimental nonselective broth enrichment (NSB) protocol and variations of the standard cold-enrichment (CE) protocol for the recovery of heat-injured Listeria monocytogenes. Bacterial cells (10(7)/ml) were suspended in sterile milk and heated at 71.7 degrees C in a slug-flow heat exchanger for holding times ranging from 1 to 30 s. Surviving cells were determined (50% endpoint) by the given protocols, and the following D values were obtained: NSB, D = 2.0 +/- 0.5 s; FDA, D = 1.4 +/- 0.3 s; USDA, D = 0.6 +/- 0.2 s; CE, D less than or equal to 1.2 s. The respective direct-plating media used in these enrichments were also analyzed for recovery, and the following D values were calculated from the enumeration of surviving cells; NSB, D = 2.7 +/- 0.8 s; FDA, D = 1.3 +/- 0.4 s; USDA, D = 0.7 +/- 0.2 s. The low levels of heat-injured L. monocytogenes cells which were detected at inactivation endpoints on the optimal nonselective media (25 degrees C for 7 days) failed to recover and multiply during experimental CEs (4 degrees C for 28 days). Initial inactivation experiments in which raw whole milk was used as the heating menstruum gave much lower recoveries with all protocols. The detectable limits for uninjured cells that were suspended in raw milk were similar (0.35 to 3.2 cells per ml) for the standard CE, FDA, and USDA protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of modified-atmosphere storage on the growth of uninjured and heat-injured Aeromonas hydrophila

The growth of uninjured and heat-injured Aeromonas hydrophila incubated at 5 degrees C (22 days) and 30 degrees C (31 h) under air, N2, and CO2 was investigated. At 30 degrees C, the growth patterns of cells on brain heart infusion agar incubated under air and N2 were similar, although slight differences in the lengths of the lag phases and the final populations were detected. The lag phases of cells incubated under air and N2 were substantially longer at 5 degrees C than at 30 degrees C. The population of uninjured A. hydrophila incubated at 5 degrees C under air and N2 remained constant, whereas the number of injured cells declined before the exponential growth phase. Growth at 5 degrees C was enhanced when uninjured and heat-injured A. hydrophila were incubated under N2. At 30 degrees C, cells incubated under CO2 exhibited noticeably longer lag phases and lower growth rates than did cells incubated under air and N2. The viable populations of uninjured and heat-injured cells incubated at 5 degrees C under CO2 declined steadily throughout incubation.     

Influence of modified-atmosphere storage on the growth of uninjured and heat-injured Aeromonas hydrophila.

The growth of uninjured and heat-injured Aeromonas hydrophila incubated at 5 degrees C (22 days) and 30 degrees C (31 h) under air, N2, and CO2 was investigated. At 30 degrees C, the growth patterns of cells on brain heart infusion agar incubated under air and N2 were similar, although slight differences in the lengths of the lag phases and the final populations were detected. The lag phases of cells incubated under air and N2 were substantially longer at 5 degrees C than at 30 degrees C. The population of uninjured A. hydrophila incubated at 5 degrees C under air and N2 remained constant, whereas the number of injured cells declined before the exponential growth phase. Growth at 5 degrees C was enhanced when uninjured and heat-injured A. hydrophila were incubated under N2. At 30 degrees C, cells incubated under CO2 exhibited noticeably longer lag phases and lower growth rates than did cells incubated under air and N2. The viable populations of uninjured and heat-injured cells incubated at 5 degrees C under CO2 declined steadily throughout incubation.     

Pyrolysis gas chromatography of Streptococcus faecalis: effect of cultural conditions on pyrochromatograms

Streptococcus faecalis 251 was cultured under a variety of different growth conditions, i.e. incubation for 24 or 70 h; at 22 degrees, 37 degrees or 45 degrees C; on blood agar or on MacConkey agar plates; aerobically or anaerobically. Replicate cultures were analysed by pyrolysis-gas liquid chromatography on columns of 7% Carbowax 2 M, TPA on Chromosorb G (AW-DMCS, 80-100 mesh) programmed from 40 degrees C up to 170 degrees C. Culture grown under identical conditions resulted in reproducible pyrochromatograms which were only slightly modified by change in temperature of growth from 37 degrees to 45 degrees C, or length of growth from 24 to 70 h, or growth on MacConkey agar instead of blood agar. Growth under anaerobic conditions resulted in a modified pyrochromatogram; while growth at only 22 degrees C resulted in a major change in pyrochromatogram.     

Agar underlay method for recovery of sublethally heat-injured bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0. 05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60 degrees C for 1.5 min in buffer or 80 degrees C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens. It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus. It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43 degrees - 45 degrees C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10-20 micrograms/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens. The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfrigens could be obtained by pre-incubation at 37 degrees C of inoculated media for 2-4 h followed by overnight incubation at 43 degrees - 45 degrees C. Tryptose-sulphite-cycloserine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens. This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus. Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clostridia on BCP is presented.     

Colony formation by sublethally heat-injured Zygosaccharomyces rouxii as affected by solutes in the recovery medium and procedure for sterilizing medium

Recovery and colony formation by healthy and sublethally heat-injured cells of Zygosaccharomyces rouxii as influenced by the procedure for sterilizing recovery media (YM agar [YMA], wort agar, cornmeal agar, and oatmeal agar) were investigated. Media were supplemented with various concentrations of glucose, sucrose, glycerol, or sorbitol and sterilized by autoclaving (110 degrees C, 15 min) and by repeated treatment with steam (100 degrees C). An increase in sensitivity was observed when heat-injured cells were plated on glucose-supplemented YMA at an aw of 0.880 compared with aws of 0.933 and 0.998. Colonies which developed from unheated and heated cells on YMA at aws of 0.998 and 0.933 generally exceeded 0.5 mm in diameter within 3.5 to 4 days of incubation at 25 degrees C, whereas colonies formed on YMA at an aw of 0.880 typically did not exceed 0.5 mm in diameter until after 5.5 to 6.5 days of incubation. The number of colonies exceeding 0.5 mm in diameter which were formed by heat-injured cells on YMA at an aw of 0.880 was 2 to 3 logs less than the total number of colonies detected, i.e., on YMA at an aw of 0.933 and using no limits of exclusion based on colony diameter. A substantial portion of cells which survived heat treatment were sublethally injured as evidenced by increased sensitivity to a suboptimum aw (0.880). In no instance was recovery of Z. rouxii significantly affected by medium sterilization procedure when glucose or sorbitol was used as the aw-suppressing solute.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colony formation by sublethally heat-injured Zygosaccharomyces rouxii as affected by solutes in the recovery medium and procedure for sterilizing medium.

Recovery and colony formation by healthy and sublethally heat-injured cells of Zygosaccharomyces rouxii as influenced by the procedure for sterilizing recovery media (YM agar [YMA], wort agar, cornmeal agar, and oatmeal agar) were investigated. Media were supplemented with various concentrations of glucose, sucrose, glycerol, or sorbitol and sterilized by autoclaving (110 degrees C, 15 min) and by repeated treatment with steam (100 degrees C). An increase in sensitivity was observed when heat-injured cells were plated on glucose-supplemented YMA at an aw of 0.880 compared with aws of 0.933 and 0.998. Colonies which developed from unheated and heated cells on YMA at aws of 0.998 and 0.933 generally exceeded 0.5 mm in diameter within 3.5 to 4 days of incubation at 25 degrees C, whereas colonies formed on YMA at an aw of 0.880 typically did not exceed 0.5 mm in diameter until after 5.5 to 6.5 days of incubation. The number of colonies exceeding 0.5 mm in diameter which were formed by heat-injured cells on YMA at an aw of 0.880 was 2 to 3 logs less than the total number of colonies detected, i.e., on YMA at an aw of 0.933 and using no limits of exclusion based on colony diameter. A substantial portion of cells which survived heat treatment were sublethally injured as evidenced by increased sensitivity to a suboptimum aw (0.880). In no instance was recovery of Z. rouxii significantly affected by medium sterilization procedure when glucose or sorbitol was used as the aw-suppressing solute.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice

Studies were done to determine the interacting effects of pH, NaCl, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5 degrees C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5 degrees C. Growth was observed in heat-sterilized unclarified cabbage juice containing less than or equal to 5% NaCl and tryptic phosphate broth containing less than or equal to 10% NaCl. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58 degrees C (pH 5.6), 4 X 10(6) cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCl; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heat-stressed cells at 5 degrees C for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5 degrees C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present.     

Improved Isolation of Anaerobic Bacteria From the Gingival Crevice Area of Man

A roll tube technique (Hungate method) was employed in an attempt to cultivate a maximal portion of the organisms in the gingival crevice area of man. This technique achieves an anaerobic state by flushing the local environment with oxygen-free gas. Once collected, the crevicular debris was immediately placed into sterile oxygen-free test tubes which were flushed out by the oxygen-free gas. In this manner, the samples were weighed, dispersed, diluted, and cultured in roll tubes and plates. The medium for control (Brewer Jar technique) and Hungate techniques was Heart Infusion Agar fortified with 10% defibrinated horse blood. When the Hungate technique was used, the recovery of viable bacteria, as a percentage of the direct microscopic count, was significantly greater than plates incubated aerobically or utilizing the Brewer Anaerobic technique. Cultural counts by using the Hungate method averaged 41.3% for six samples when 90% nitrogen and 10% hydrogen were used, 70.4% for eight samples when 85% nitrogen, 10% hydrogen, and 5% carbon dioxide were used, and 63.4% for eight samples when 100% carbon dioxide was the gaseous atmosphere. At no time were cultural counts, by using anaerobic plates (Brewer Jar), more than 24% of the direct microscopic count. This suggests that exclusion of oxygen and the presence of carbon dioxide maximized recovery of gingival crevice bacteria.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

Thermal inactivation of Listeria monocytogenes during rapid and slow heating in sous vide cooked beef

T.B. HANSEN AND S. KNØCHEL. 1996. Heating at slowly rising temperatures is suspected to enhance thermotolerance in Listeria monocytogenes and, since anaerobic environments have been shown to facilitate resuscitation of heat-injured cells of this micro-organism, concern may arise about the possibility of L. monocytogenes surviving in minimally preserved products. The effect of rapid (> 10°C min^-1) and slow (0.3 and 0.6°C min^-1) heating on survival of L. monocytogenes in sous vide cooked beef was therefore examined at mild processing temperatures of 56, 60 and 64°C. No statistically significant difference ( P = 0.70) was observed between the tested heating regimes. Since the average pH of beef was low (5.6), and little or no effect was observed, a pH-dependency of heat shock-induced thermotolerance in L. monocytogenes is suggested to account for this result.