Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.     

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.     

Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage

Direct plating, selective enrichment, and cold enrichment followed by secondary selective enrichment procedures were compared for detecting and enumerating Listeria monocytogenes in chopped cabbage stored at 5 degrees C for up to 64 days. Addition of Fe3+ to solid media enhanced detection of the organism. Cold enrichment (5 degrees C) in nutrient broth and brain heart infusion broth followed by secondary enrichment (48 h, 30 degrees C) in Trypticase soy-yeast extract-antibiotic broth and thiocyanate-nalidixic acid broth and plating on selective agar media (Doyle and Schoeni selective enrichment agar [minus acriflavin hydrochloride, supplemented with 5 micrograms of Fe3+/ml] and McBride Listeria agar) resulted in the detection of highest populations.     

Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage.

Direct plating, selective enrichment, and cold enrichment followed by secondary selective enrichment procedures were compared for detecting and enumerating Listeria monocytogenes in chopped cabbage stored at 5 degrees C for up to 64 days. Addition of Fe3+ to solid media enhanced detection of the organism. Cold enrichment (5 degrees C) in nutrient broth and brain heart infusion broth followed by secondary enrichment (48 h, 30 degrees C) in Trypticase soy-yeast extract-antibiotic broth and thiocyanate-nalidixic acid broth and plating on selective agar media (Doyle and Schoeni selective enrichment agar [minus acriflavin hydrochloride, supplemented with 5 micrograms of Fe3+/ml] and McBride Listeria agar) resulted in the detection of highest populations.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

Method for the detection of injured Vibrio parahaemolyticus in seafoods.

The sensitivity of Vibrio parahaemolyticus cells to refrigeration and frozen storage and the development of a method for detecting injured and uninjured V. parahaemolyticus cells were studied. Cell suspensions in different kinds of seafood homogenates were either regrigerated (4 degrees C) or frozen (-20 degrees C), stored, and examined for cell survival during storage. V. parahaemolyticus cells were sensitive to both storage temperatures. Many cells died, and many survivors were sublethally injured. In general, refrigeration storage appeared to be more injurious than frozen storage. The initial recovery of the sublethally injured cells was highest in a nutritionally rich, nonselective liquid medium such as Trypticase soy broth, whereas maximum cell multiplication was observed in Trypticase soy broth containing 3% NaCl. The sublethally injured V. parahaemolyticus cells demonstrated sensitivity to the selective enrichment medium, glucose salt teepol broth. From these findings, a new method (designated as the "repair-detection" method) was developed for the isolation and enumeration of V. parahaemolyticus. Comparative studies between the recommended and the repair-detection methods showed that injured V. parahaemolyticus cells were present in commercial seafoods and that the repair-detection method was definitely more effective for the detection of total numbers of V. parahaemolyticus cells.     

Radiation resistance and injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25 degrees C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and -30 degrees C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at -20 degrees C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at -20 degrees C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at -20 degrees C, nor did storage at -20 degrees C alter the cell's resistance to irradiation at 25 degrees C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36 degrees C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36 degrees C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5 degrees C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36 degrees C for 1 day than at 5 degrees C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Relationships of Temperature and Sodium Chloride Concentration to the Survival of Vibrio parahaemolyticus in Broth and Fish Homogenate

The interaction of temperature and NaCl concentration in affecting the survival of three strains of Vibrio parahaemolyticus was studied in Trypticase soy broth and fish homogenate. Cells of V. parahaemolyticus suspended in Trypticase soy broth without NaCl were quite unstable and readily killed. The presence of NaCl appeared to be protective to the cells at 48 +/- 1 C, with the optimal concentration strain-dependent for the 3 to 12% range tested. Temperatures of 5 +/- 1, -5 +/- 1, and -18 +/- 1 C reduced the number of viable organisms per milliliter regardless of the NaCl concentration. In the presence of NaCl, viable cells, in numbers ranging up to 580 per ml, were still detected at the end of 30 days of storage. Similar results were obtained for cells suspended in fish homogenate, except that fish homogenate itself was protective as compared with Trypticase soy broth. This protection was significantly lower than that provided by NaCl in any amount tested.     

Alkalotolerance of Yersinia enterocolitica as a basis for selective isolation from food enrichments.

Alkalotolerance of Yersinia enterocolitica measured in solutions of potassium hydroxide with 0.5% sodium chloride was influenced by the cell suspension medium, temperature, and growth phase. The rate of cell destruction (delta log N per minute) was five times greater at 30 degrees C than at 20 degrees C. Differences in the degree of cell destruction at various concentrations of potassium hydroxide were related to pH and not to osmolarity. The addition of peptones to potassium hydroxide provided a protective effect that was greater for cells suspended in Trypticase soy broth than for those suspended in phosphate-buffered sorbitol-bile salts broth. Log-phase cells were less alkalotolerant than cells in the stationary phase of growth. A modified procedure for alkali treatment, using peptone-supplemented 0.5% potassium hydroxide-0.5% sodium chloride and the addition of a pH 6.6 buffer after treatment to prevent further cell destruction, was used to observe a marked difference in alkalotolerance between Y. enterocolitica and other gram-negative bacteria. Despite this difference, alkali treatment was not highly successful for recovery of Y. enterocolitica from enrichments of seeded foods in comparison with selective enrichment in bile-oxalate-sorbose broth.     

A comparative study of preservation and storage of Haemophilus influenzae

The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20 degrees C and -70 degrees C. Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20 degrees C and -70 degrees C from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20 degrees C. Initial concentration did not influence survival after storage at -70 degrees C. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.     

Modeling and predicting the simultaneous growth of Escherichia coli O157:H7 and ground beef background microflora for various enrichment protocols

The simultaneous growth of Escherichia coli O157:H7 (O157) and the ground beef background microflora (BM) was described in order to characterize the effects of enrichment factors on the growth of these organisms. The different enrichment factors studied were basal medium (Trypticase soy broth and E. coli broth), the presence of novobiocin in the broth, and the incubation temperature (37 degrees C or 40 degrees C). BM and O157 kinetics were simultaneously fitted by using a competitive growth model. The simple competition between the two microfloras implied that O157 growth stopped as soon as the maximal bacterial density in the BM was reached. The present study shows that the enrichment protocol factors had little impact on the simultaneous growth of BM and O157. The selective factors (i.e., bile salts and novobiocin) and the higher incubation temperature (40 degrees C) did not inhibit BM growth, and incubation at 40 degrees C only slightly improved O157 growth. The results also emphasize that when the level of O157 contamination in ground beef is low, the 6-h enrichment step recommended in the immunomagnetic separation protocol (ISO EN 16654) is not sufficient to detect O157 by screening methods. In this case, prior enrichment for approximately 10 h appears to be the optimal duration for enrichment. However, more experiments must be carried out with ground beef packaged in different ways in order to confirm the results obtained in the present study for non-vacuum- and non-modified-atmosphere-packed ground beef.     

Effect of environmental conditions during heating on commercial spore strip performance.

Commercial biological indicator spore strips in glassine envelopes, produced by three manufacturers, were evaluated by fraction-negative procedures after being heated at 121.0 +/- 0.05 degrees C. Only one type of spore strip met the manufacturer's specifications. The strips of one manufacturer were further evaluated by fraction-negative and survivor curve-plate count procedures after being heated under several conditions (enclosed in glassine envelopes, in trypticase soy broth plus 0.0015% bromocresol purple, in Trypticase soy broth alone in Water for Injection, directly); Trypticase soy broth plus bromocresol purple and tryptic soy agar, respectively, were used as recovery media. The heating condition affected the D-value of the spore strip. Recovery procedures also had an effect; in all cases, the D-values obtained from the survivor curve tests were larger than those obtained from fraction-negative tests carried out under the same conditions. To determine if the differences in D-values between the two evaluation procedures were caused by the recovery media, we evaluated, by both methods, one type of spore strip heated directly and in glassine envelopes, using tryptic soy agar plus bromocresol purple and Trypticase soy broth plus 1.5% agar, respectively, as the recovery media. The survivor curve results showed that for both enclosed and unenclosed spore strips, there was a marked difference between the two recovery media; however, there was no difference when fraction-negative tests were used.     

Listeria monocytogenes Scott A: cell surface charge, hydrophobicity, and electron donor and acceptor characteristics under different environmental growth conditions

We determined the variations in the surface physicochemical properties of Listeria monocytogenes Scott A cells that occurred under various environmental conditions. The surface charges, the hydrophobicities, and the electron donor and acceptor characteristics of L. monocytogenes Scott A cells were compared after the organism was grown in different growth media and at different temperatures; to do this, we used microelectrophoresis and the microbial adhesion to solvents method. Supplementing the growth media with glucose or lactic acid affected the electrical, hydrophobic, and electron donor and acceptor properties of the cells, whereas the growth temperature (37, 20, 15, or 8 degrees C) primarily affected the electrical and electron donor and acceptor properties. The nonlinear effects of the growth temperature on the physicochemical properties of the cells were similar for cells cultivated in two different growth media, but bacteria cultivated in Trypticase soy broth supplemented with 6 g of yeast extract per liter (TSYE) were slightly more hydrophobic than cells cultivated in brain heart infusion medium (P < 0.05). Adhesion experiments conducted with L. monocytogenes Scott A cells cultivated in TSYE at 37, 20, 15, and 8 degrees C and then suspended in a sodium chloride solution (1.5 x 10(-1) or 1.5 x 10(-3) M NaCl) confirmed that the cell surface charge and the electron donor and acceptor properties of the cells had an influence on their attachment to stainless steel.     

Pasteurization of salted whole egg inoculated with Arizona or Salmonella.

Recently, Arizona bacteria, close relatives of Salmonella, were recovered from salted whole egg that had been pasteurized by the presently recommended process of 63.3 degrees C (146 degrees F) for 3.5 min. Because of this and the fact that the heat resistance of Arizona in salted whole egg had not been determined, the present study was undertaken. Arizona or Salmonella, grown in Trypticase soy broth supplemented with 2% yeast extract in Fernbach flasks covered with aluminum foil over cotton and guaze at 35 degrees C with shaking at 176 rpm for about 96 h, were found to have the greatest degree of heat resistance. As expected, these cells, when inoculated into salted whole egg at 10(7) cells per ml, survived heating at 63.3 degrees C (146 degrees F) for 3.5 min in a two-phase slug flow heat exchanger. To consistently achieve a 7-log kill of typical Salmonella or Arizona, a treatment of 67 degrees C (152.6 degrees F) for 3.5 min was required. However, if a 7-log kill is mandatory, it remains to be determined whether this process affect the functional properties of this product.     

Pasteurization of salted whole egg inoculated with Arizona or Salmonella.

Recently, Arizona bacteria, close relatives of Salmonella, were recovered from salted whole egg that had been pasteurized by the presently recommended process of 63.3 degrees C (146 degrees F) for 3.5 min. Because of this and the fact that the heat resistance of Arizona in salted whole egg had not been determined, the present study was undertaken. Arizona or Salmonella, grown in Trypticase soy broth supplemented with 2% yeast extract in Fernbach flasks covered with aluminum foil over cotton and guaze at 35 degrees C with shaking at 176 rpm for about 96 h, were found to have the greatest degree of heat resistance. As expected, these cells, when inoculated into salted whole egg at 10(7) cells per ml, survived heating at 63.3 degrees C (146 degrees F) for 3.5 min in a two-phase slug flow heat exchanger. To consistently achieve a 7-log kill of typical Salmonella or Arizona, a treatment of 67 degrees C (152.6 degrees F) for 3.5 min was required. However, if a 7-log kill is mandatory, it remains to be determined whether this process affect the functional properties of this product.     

Repair of Injury Induced by Freezing Escherichia coli as Influenced by Recovery Medium

Freezing an aqueous suspension of Escherichia coli NCSM at -78 C for 10 min, followed by thawing in water at 8 C for 30 min, resulted in the death of approximately 50% of the cells, as determined by their inability to form colonies on Trypticase soy agar containing 0.3% yeast extract (TSYA). Among the survivors, more than 90% of the cells were injured, as they failed to form colonies on TSYA containing 0.1% deoxycholate. Microscope counts and optical density determinations at 600 nm suggested that death from freezing was not due to lysis of the cells. Death and the injury were accompanied by the loss of 260- and 280-nm absorbing materials from the intracellular pool. Injury was reversible as the injured cells repaired in many suitable media. The rate of repair was rapid and maximum in a complex nutrient medium such as Trypticase soy broth supplemented with yeast extract. However, inorganic phosphate, with or without MgSO₄, was able to facilitate repair. Repair in phosphate was dependent on the pH, the temperature, and the concentration of phosphate.     

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.     

Survival and growth characteristics of Escherichia coli associated with hemorrhagic colitis.

Escherichia coli O157:H7 in ground beef was more sensitive to heat than salmonellae, but survived for 9 months at -20 degrees C with little change in number. The organisms grew well in Trypticase soy broth (BBL Microbiology Systems) between 30 and 42 degrees C, with 37 degrees C being optimal for growth. E. coli O157:H7 grew poorly in the temperature range (44 to 45.5 degrees C) generally used for recovery of E. coli from foods.     

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.