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J APPL POULT RES 2009. 18:297-309. doi:10.3382/japr.2008-00109
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Research Reports

Evaluation of plating media for recovering Salmonella from thermally treated egg albumen1

J. B. Gurtler2

Food Safety Intervention Technologies Research Unit, USDA, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038-8551

2 Corresponding author: joshua.gurtler{at}ars.usda.gov


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Salmonella spp. present in pasteurized egg albumen are often difficult to recover by direct plating because of thermal injury and the presence of innate iron binding and other antimicrobials in egg white. The literature has reported a multiplicity of selective and nonselective media used to recover heat-injured Salmonella, to measure the proportion of injured cells, or both. This study compared the proficiency of selective and nonselective plating media for supporting colony development or for assessing bacterial injury of heat-stressed Salmonella from egg albumen. A 6-strain composite of Salmonella was added to albumen (pH 9.0), heated at 53.3°C for 3.1 min, and plated on 26 nonselective and 22 selective media. Recovery of heat-injured salmonellae varied little (≤0.52 log cfu/mL) among the nonselective media tryptic soy agar, plate count agar, dextrose tryptone agar, or brain heart infusion agar, regardless of the manufacturer. Selective media that were optimal for recovery of salmonellae from albumen included 3 brilliant green agars, Levine eosin methylene blue agar, and bismuth sulfite agar, which recovered more cells (P ≤ 0.05) than selenite-cystine, tetrathionate, xylose-lysine-tergitol-4 , xylose lysine deoxycholate, or Rappaport-Vassiliadis agars. The results of this study may assist in choosing selective or nonselective media to maximize the recovery of Salmonella from thermally treated albumen.

Key Words: egg albumen • Salmonella • medium • pasteurization • recovery


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
The USDA National Agricultural Statistics Service reported that US egg companies produced 2.61 billion pounds of liquid egg in 2007 from 24 billion eggs [1]. The Egg Products Inspection Act of 1970 regulates egg products through the USDA Food Safety and Inspection Service (FSIS) by mandating minimum thermal pasteurization requirements for specified egg products. These standards were based on data for the inactivation of Salmonella in liquid egg products acquired before 1970 and are currently being reevaluated in light of recent risk assessments, which take into account changes in industrial practices such as in-line egg processing and variations in egg product formulations [2, 3]. Regulations may be amended in accordance with Hazard Analysis and Critical Control Point and Sanitation Standard Operating Procedure compliance guidelines so as to incorporate lethality-based performance standards [4].

Pasteurized egg products have not been recognized as a source of human illness in the United States since the Egg Products Inspection Act of 1970 was issued. However, the USDA FSIS has concluded that current process standards are not completely effective in eliminating Salmonella from all egg products, and predictive models estimate that up to 5,500 people per year may contract salmonellosis from pasteurized egg products [2, 5]. The FSIS reported that during routine USDA surveillance, Salmonella was recovered from 0.47% of pasteurized egg products between the years of 1998 and 2003 [4]. The USDA FSIS risk assessment for Salmonella in shell eggs reported that current minimum pasteurization standards impart 5.9, 5.5, and 3.3 log10 reductions of Salmonella in liquid whole egg, liquid egg yolk, and liquid egg white (albumen), respectively [2]. They estimated that if all liquid egg whites in the United States were pasteurized for a 6-log10 reduction of Salmonella, the annual number of human illnesses from pasteurized egg albumen products could be reduced from 2,600 to 270 per year.

Problems relating to recovering Salmonella from egg products containing yolk have been reported [6–8], although recovery of Salmonella from albumen has proven most difficult [9, 10]. Innate albumen antimicrobials (e.g., ovotransferrin, lysozyme, avidin, etc.) [3, 11] may compound injury that Salmonella incurs during pasteurization, complicating bacterial recovery and enumeration. Studies that are performed to assess the presence and levels of Salmonella in pasteurized liquid egg products by direct plating require the use of media with a high level of sensitivity that will maximize resuscitation and enumeration of thermally injured bacteria. Non-selective plating media that have been used to recover Salmonella in various states of injury by direct plating include brain heart infusion agar [9, 12], solidified buffered peptone water, solidified M9 minimal salts broth, solidified tryptic soy broth [13], solidified Dey-Engley neutralizing medium [14], plate count agar [15], and tryptic soy agar [16]. Selective plating media that have been used to recover Salmonella in various states of injury by direct plating include tryptic soy agar + 0.6% yeast extract with 5% sodium chloride [17], bismuth sulfite agar [18], xyloselysine-tergitol-4 agar [18, 19], solidified selenite broth, solidified tetrathionate broth [20], Levine eosin methylene blue agar with 2% sodium chloride [21], MacConkey sorbitol agar [22], xylose lysine deoxycholate agar, brilliant green agar [10], selenite-cystine agar, Rappaport-Vassiliadis agar [23], Hektoen enteric agar, Rambach Plus agar, and modified lysine iron agar [24].

Reactive oxygen species (ROS) that are present in plating media impede the resuscitation of injured bacteria. Hydroxyl radicals and other ROS produced during autoclave-sterilization of media, or shortly thereafter, are highly toxic to bacterial DNA, membrane lipids, and cellular proteins [25]; thus, various oxygen tension-reducing compounds have been added to selective and nonselective media to enhance the recovery of injured bacteria [26].

This study was designed to assess the relative proficiency of 22 selective and 26 nonselective plating media in colony development of a 6-strain composite of Salmonella from thermally treated albumen to select media that will maximize the recovery of heat-injured Salmonella or indicate the level of injury for future egg-pasteurization studies. An investigation of selective compounds (NaCl, KMnO4, EDTA, sodium deoxycholate, and calcium hypochlorite) added to tryptic soy agar was also undertaken to determine their inhibiting or resuscitating effects on the Salmonella composite and to assess their potential for use in future experiments.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Bacterial Strains and Preparation of Cultures for Heat Treatment
Six strains [27] of Salmonella enterica were examined for their response to thermal stress in liquid egg white and recovery on 26 nonselective and 22 selective media. Cells were grown for 24 h in a nutritive broth and were concentrated 10-fold [28].

Preparation of Egg Albumen and Recovery Media
Albumen was prepared [29] and inoculated with a 6-strain composite of salmonellae to 7.74 log cells/mL. All media (Tables 1Go and 2Go) were purchased in dehydrated form and prepared according to the manufacturers’ specifications [30] unless noted otherwise in the References and Notes.


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  		Table 1. Nonselective media used to recover a 6-strain composite of Salmonella from egg albumen heated at 53.3°C for 3.1 min
 

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  		Table 2. Selective media used to recover a 6-strain composite of Salmonella from egg albumen heated at 53.3°C for 3.1 min
 
Thermal Treatment of Inoculated Albumen in Thermal-Death-Time Disks
Inoculated albumen was thermally treated by means of aluminum thermal-death-time (TDT) disks, which have been described by Jin et al. [31]. Sterile TDT disks were loaded with 0.9 mL of inoculated egg white, hermetically sealed, and immersed in a recirculating water bath [32] at 53.3°C for 3.1 min. This treatment was optimized in preliminary tests (data not shown) to effect an approximate 2-log reduction in population and to induce thermally mediated bacterial injury to cells in the surviving population. Temperatures were monitored with type K thermocouples and electronically recorded [33]. One thermocouple was placed directly in the center of the water bath to measure ambient water temperature. A second thermocouple was affixed inside a TDT disk through a hole drilled in the bottom of the disk, sealed with a waterproof adhesive, and filled with 0.9 mL of uninoculated albumen to monitor the heating time and treatment temperature for each experiment. Heating times began when the internal temperature of the TDT disk reached within 0.5°C (52.8°C) of the target temperature (53.3°C). After the thermal treatment, disks were immediately immersed in a slush ice bath to arrest the thermal effects on bacterial cells.

Plating of Thermally Treated Cells on Selective and Nonselective Media and Measurement of pH and Oxidation-Reduction Potential
The TDT disks were surface-sanitized by immersion in 70% ethanol, rinsed with sterile deionized water, and opened aseptically, and then 0.7 mL of the heated albumen was removed with a pipette and added to 6.3 mL of 0.1% peptone solution. Serial dilutions of thermally treated albumen were spiral plated [34] in duplicate on the surface of nonselective or selective agar. All plates were incubated at 37°C for 48 h and colonies were counted [35]. The pH and oxidation-reduction potential (ORP) of each medium and albumen sample were measured [36] in duplicate.

Statistical Analysis
Three replicate trials were conducted, and duplicate samples were averaged before statistical analysis. Data were analyzed by ANOVA using SAS software version 9.1 [37]. Significant differences (P ≤ 0.05) in the recovery of Salmonella within selective or nonselective media were determined by LSD.


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Heating and Cooling Times
The heating time needed to increase samples from 21°C to within 0.5°C of the target temperature (53.3°C) was 40 s, and samples were held for an additional 3.1 min. The cooling times needed to lower the temperature from 53.3°C to 15.0 and 0°C were 12 and 55 s, respectively.

Results of pH and ORP Measurements
After inoculation, the average pH of albumen was 9.0 and the average ORP measurement was 114 mV. There were few differences in pH and ORP measurements within selective and nonselective media (Tables 3Go and 4Go). All nonselective media pH values were between 6.31 and 7.55, with ORP readings between 170 and 263 mV, except for presterilization and poststerilization Dey-Engly neutralizing broth, which had pH values of 8.03 and 8.37 and had ORP readings of 80 and 114 mV, respectively. Selective media pH readings were between 6.40 and 7.76, except for Rappaport-Vassiliadis broth and tryptic soy agar + 0.1% KMnO4, which had pH values of 5.34 and 8.37, respectively. Selective media ORP values were between 129 and 235, except for tetrathionate broth and overheated bismuth sulfite agar, with ORP readings of 82 and 104 mV, respectively. Overall, differences in pH and ORP values did not appear to be a major factor in the proficiency of media to recover injured cells. All media had ORP and pH values within the ranges reported for supporting the growth of Salmonella [38–40].


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  		Table 3. Oxidation-reduction potential (ORP) and pH of nonselective media used to recover a 6-strain composite of Salmonella from egg albumen heated at 53.3°C for 3.1 min
 

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  		Table 4. Oxidation-reduction potential (ORP) and pH of selective media at 22°C used to recover a 6-strain composite of Salmonella from egg albumen heated at 53.3°C for 3.1 min
 
Population of Salmonella in Inocula and Albumen Before Heating
Average populations of inocula were 9.94 log cfu/mL for the 6 individual strains and 9.98 log cfu/mL for the 6-strain composite. No mesophilic aerobic microorganisms were recovered from uninoculated albumen samples. Inoculated albumen contained an average of 7.74 log cells/ mL of Salmonella before heating.

Recovery of Heat-Stressed Salmonella from Thermally Treated Albumen on Nonselective Agar
Mean composite values from direct plating on nonselective agar were analyzed to determine significant differences in the numbers of Salmonella recovered on each medium, and are separated into 6 groupings to simplify discussion (Figure 1Go). Group I, which included tryptic soy agar + 0.01 EDTA, plate count agar (company A), tryptic soy agar, dextrose tryptone agar, pre-sterilization nutrient broth, and presterilization universal preenrichment broth, recovered the greatest numbers of heat-stressed Salmonella, although only tryptic soy agar + 0.01% EDTA recovered significantly more Salmonella than the media in group II, which included poststerilization universal preenrichment broth, post-sterilization nutrient broth, and presterilization lactose broth. The chelating agent EDTA, at sub-bacteriostatic levels, may provide protection to injured cells by sequestering iron to prevent ROS generation. Facon and Skura [41] reported that the numbers of Salmonella Enteritidis held for 4 h at 37°C in tryptic soy broth with 25 mM EDTA did not decline when compared with holding in tryptic soy broth without EDTA. The presence of lethal ROS in agar may be related to metal-catalyzed hydroxyl production, which is enhanced after high-temperature sterilization. Fenton-mediated hydroxyl radical formation occurs when hydrogen peroxide (generated in microbiological media during heating) initiates the conversion of ferrous iron (Fe2+) to ferric iron (Fe3+) [42, 43]. Hydroxyl radicals are the most reactive oxygen radical and are known to oxidize lipids, nucleic acids, and proteins [44]. Chelators can block the generation of hydroxyls by sequestering unbound iron [45]. McFeters and Singh [46] reported that varying medium compositions and incubation temperatures as well as the added chelating compounds were all factors in the recovery of injured bacteria.


Figure 1
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  		Figure 1. Populations of a 6-strain composite of Salmonella inoculated into egg albumin, heated for 3.1 min at 53.3°C, recovered on 26 nonselective plating media. Values represent mean log10 cfu/mL of 3 replicate experiments. Mean values that are not followed by the same letter are significantly different (P ≤ 0.05). Error bars represent SD.

 
The numbers of Salmonella recovered on dextrose tryptone agar were equal to those on all media in group I. Dextrose tryptone agar could be useful in recovering injured Salmonella as well as in differentiating changes in pH because it contains bromcresol purple, a pH indicator. Solidified universal preenrichment broth, although containing sodium pyruvate, which is known to enhance the recovery of heat-injured bacteria, recovered no greater numbers of Salmonella than did plate count agar (company A), tryptic soy agar, or dextrose tryptone agar, which contained no sodium pyruvate. The use of solidified enrichment broths (lactose broth, Luria-Bertani broth, buffered peptone water, buffered peptone water #2, tryptic soy broth, brain heart infusion broth, and nutrient broth) imparted no benefit in recovering Salmonella when compared with group I media or other commonly used nonselective recovery media evaluated. Salmonella recovery differed little ( 0.52 log cfu/mL) among varying brands of the nonselective media tryptic soy agar, plate count agar, dextrose tryptone agar, or brain heart infusion agar.

No significant differences in the recovery of Salmonella occurred when granulated agar was added to nonselective media before autoclave sterilization rather than when melted autoclave-sterilized agar was added to filter-sterilized broths (i.e., poststerilization universal preenrichment broth, poststerilization nutrient broth, poststerilization lactose broth, poststerilization Luria-Bertani broth, poststerilization tryptic soy broth, poststerilization buffered peptone water #2, and poststerilization Dey-Engly neutralizing broth) before dispensing into Petri dishes. Four media in group I (tryptic soy agar + 0.01% EDTA, plate count agar [company A], tryptic soy agar, and dextrose tryptone agar) recovered greater numbers of Salmonella than other media reported to have been used to recover injured Salmonella (i.e., brain heart infusion agar, buffered peptone water, presterilization tryptic soy broth, presterilization Dey-Engly neutralizing broth, plate count agar [company A], and plate count agar [company B]). Augustin and Carlier [47] reported that culture medium type and manufacturer significantly affected the recovery of Enterobacteriaceae. Stephens et al. [44] demonstrated a greater than 1,000-fold difference in heat-stressed Salmonella Typhimurium recovered among peptone from 12 sources.

Although there have been reports of injured Salmonella recovering in higher numbers on a minimal medium than on tryptic soy agar, fewer cells in this study (4.87 log cfu/mL) were recovered on Davis minimal agar (group V) than on media in groups I to IV (5.29 to 5.94 log cfu/ mL). Mackey and Derrick [20] recovered equivalent numbers of healthy Salmonella Typhimurium LT2 on nutrient agar and solidified M9 minimum salts broth (7.85 cfu/mL), although after heating at 52°C for 90 min in phosphate buffer, populations of Salmonella recovered were 5.93 and 5.19 log cfu/mL, respectively. Wilson and Davies [48] reported that solidified M9 minimal broth recovered greater numbers of Salmonella than tryptic soy agar with 0.5% yeast extract after cells were heated in phosphate buffer at 52°C for up to 30 min. Others have reported similar results when heat-injured cells were allowed a brief recovery period in M9 minimal broth [49]. Mackey and Derrick [50] also reported that M9 minimal agar was superior to tryptic soy agar with yeast extract for recovering cold-shocked Salmonella. They hypothesized that the differences in recovery could be attributed to lethal peroxide concentrations in tryptic soy agar with yeast extract that were measured at 12 to 30 µmol/L, whereas no peroxide was detected in M9 agar. Differences in medium composition between M9 minimal agar (which contains dipotassium phosphate, monopotassium phosphate, sodium chloride, and ammonium chloride) and Davis minimal agar (which contains contains dipotassium phosphate, monopotassium phosphate, dextrose, sodium citrate, magnesium sulfate, and ammonium sulfate) may account for the variation in the ability to recover injured Salmonella in the present study. Adding nutrients and buffers to a medium has been known to intensify substrate-accelerated bacterial death [51–54]. It has been hypothesized that autoxidation of ingredients, such as phosphate buffers and sugars, during autoclaving or shortly thereafter may lead to the formation of hydrogen peroxide and other toxic oxygen species, which would inhibit resuscitation of injured bacteria [20, 44, 50, 55–59].

Recovery of Heat-Stressed Salmonella from Thermally Treated Albumen on Selective Agar
Mean values from selective agar were analyzed to determine significant differences in Salmonella recovered on each medium and have been divided into 5 groupings to simplify discussion (Figure 2Go). Media in group I (brilliant green agar, Levine eosin methylene blue agar, brilliant green agar with phosphates, bismuth sulfite agar, and brilliant green bile agar) recovered the greatest number of injured cells among commercially available selective media in this study. Although there was no statistical difference in the recovery of Salmonella among media in group I, only brilliant green agar recovered a significantly greater number of injured Salmonella (5.84 log cfu/mL) than groups II to V (0.77 to 5.06 log cfu/mL). When raw data for Salmonella recovered on nonselective tryptic soy agar were included in the statistical analysis of Salmonella recovered on selective media, only the 8 selective media in group I recovered equivalent numbers of injured Salmonella (5.31 to 5.84 log cfu/mL) as tryptic soy agar (log 5.80 cfu/mL; Figure 1Go).


Figure 2
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  		Figure 2. Populations of a 6-strain composite of Salmonella inoculated into egg albumen, heated for 3.1 min at 53.3°C, and recovered on 22 selective plating media. Values represent mean log10 cfu/mL of 3 replicate experiments. Mean values that are not followed by the same letter are significantly different (P ≤ 0.05). Error bars represent SD.

 
Although not statistically different, fewer injured cells were recovered on overheated bismuth sulfite agar than on bismuth sulfite agar (5.05 and 5.58 log cfu/mL, respectively), demonstrating the decreased sensitivity of bismuth sulfite agar when overheated. Except for tetrathionate broth (group IV), fewer cells were recovered on xyloselysine-tergitol-4 agar than on all other commercially produced selective media or solidified selective enrichment broths, emphasizing the decreased sensitivity of xyloselysine-tergitol-4 in recovering thermally stressed salmonellae. Williams et al. [18] recovered fewer Salmonella on xyloselysine-tergitol-4 than bismuth sulfite agar from apple cider inoculated with salmonellae treated with ozone at 4 or 50°C, as well as from Salmonella-inoculated orange juice treated with ozone at 50°C.

There were no significant differences in the recovery of injured cells among varying concentrations of sodium deoxycholate added to tryptic soy agar, although there was a 0.63, 0.88, and 1.2 log cfu/mL reduction in the recovery of Salmonella on tryptic soy agar + 0.1, 0.25, and 0.4% sodium deoxycholate, respectively, when compared with the highest level of Salmonella recovered on nonselective media (5.94 log cfu/ mL; Figure 1Go). There was no significant difference in the numbers of injured Salmonella recovered on tryptic soy agar + 0.5% calcium hypochlorite or tryptic soy agar + 4.5% NaCl (1.47 and 0.77 log cfu/mL, respectively; group V) or between tryptic soy agar + 0.1% KMnO4 and Rappaport-Vassiliadis broth (4.34 and 4.07 log cfu/mL, respectively; group III). Addition of these selective compounds to media may assist in determining the degree of bacterial injury in future studies. However, further investigations would need to address the sensitivity of these compounds in recovering healthy, unstressed cells when compared with heat-injured cells. Rayman et al. [60] reported that tryptic soy agar + 0.15% KMnO4 recovered at least 100 times higher counts of heat-stressed Salmonella Senftenberg than tryptic soy agar alone, although exposure to KMnO4 at higher concentrations is known to be bactericidal [61]. Addition of 0.1% KMnO4 to tryptic soy agar in this study, however, did not enhance recovery, and at 0.5%, deterioration of media occurred during autoclaving.

Results in the present study indicate that selective and nonselective media vary in their ability to support resuscitation and colony formation by Salmonella from thermally treated albumen. Six nonselective media (tryptic soy agar + 0.01% EDTA, plate count agar [company A], tryptic soy agar, dextrose tryptone agar, presterilization nutrient broth, and presterilization universal preenrichment broth) supported recovery of the greatest, yet equivalent, numbers of Salmonella and may assist in choosing a nonselective recovery medium. This study also provides information that may be useful in choosing a commercially produced selective medium (e.g., brilliant green agar, Levine eosin methylene blue agar, brilliant green agar with phosphates, bismuth sulfite agar, and brilliant green bile agar recovered the greatest, yet equivalent, numbers of cells) to maximize the recovery of injured Salmonella after a period of resuscitation on a nonselective agar. Various studies have used methods of resuscitating injured cells by incubating on nonselective agar for a specified time, followed by exposure to a selective medium. A thin agar layer method has been used to recover heat-injured Salmonella by plating bacterial suspensions on tryptic soy agar overlaid with xylose lysine deoxycholate agar [62]. McCann et al. [63] plated heat-injured Salmonella on tryptic soy agar held at 25°C for 2 h and then overlaid with xylose lysine deoxycholate agar. The present study indicates that because 1.81, 2.58, and 3.29 log cfu/mL fewer heat-injured Salmonella were recovered on xylose lysine deoxycholate, xyloselysine-tergitol-4, and tetrathionate broth, respectively, as compared with those recovered on brilliant green agar, these media may serve as good indicators of bacterial injury. These findings may also assist in choosing a selective medium to overlay onto nonselective agar for the recovery of heat-injured Salmonella from food samples with a heterogeneous population of bacteria. In selecting an appropriate selective medium, one would also need to account for agar specificity, which would largely depend on the population and makeup of background microflora present in a food sample. The addition of ROS-reducing and other resuscitation-promoting compounds to selective or nonselective media examined in this study could further enhance the recovery of heat-injured Salmonella from liquid egg products.


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. The nonselective media tryptic soy agar, plate count agar, dextrose tryptone agar, and brain heart infusion agar all recovered maximum and equivalent numbers of heat-injured Salmonella from egg albumen.
  2. Selective media that optimized recovery of salmonellae from thermally treated albumen included 3 brilliant green agars, Levine eosin methylene blue agar, and bismuth sulfite agar.
  3. Commercially available selective media recovering the fewest cells, which may be used to assess injury of Salmonella in pasteurized albumen, were xyloselysine-tergitol-4, xylose lysine deoxycholate, selenite-cystine, tetrathionate, and Rappaport-Vassiliadis agars.


   ACKNOWLEDGMENTS
 
The author thanks Larry Beuchat (Center for Food Safety, University of Georgia, Griffin), Tony Jin, and Hyun-Gyun Yuk (USDA, Eastern Regional Research Center) for reviewing this manuscript and providing helpful feedback.


   FOOTNOTES
 
1 Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. Back


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

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  27. These strains included the type strain for Salmonella enterica serovar Enteritidis (ATCC 13076), and serovars Typhimurium (ATCC 14028, chicken isolate), Oranienburg (ATCC 9239, food poisoning outbreak), and Montevideo (ATCC 8387). Serovars Infantis F4319 and Newport H1275 are clinical isolates implicated in foodborne outbreaks, originally obtained from Patricia Griffin (Centers for Disease Control and Prevention, Atlanta, GA) and were graciously provided by Ching-Hsing Liao (USDA, Eastern Regional Research Center, Wyndmoor, PA) for use in this study.
  28. Salmonella strains were maintained on tryptic soy agar (Difco, Becton Dickinson, Sparks, MD) slants in borosilicate screw-capped test tubes at 4°C. Cultures were grown in tryptic soy broth (Difco; 10 mL) at 37°C for 24 h, with a 10-µL loop transfer made into 30 mL of tryptic soy broth and incubation at 37°C for an additional 24 h. Cells were pelleted by centrifugation at 10°C for 8 min at 14,000 x g, washed in sterile deionized water, centrifuged a second time and resuspended in 3 mL of sterile deionized water to attain a 10-fold increase in cell concentration. One milliliter from each of 6 strains was combined in a sterile test tube to create a 6-strain composite suspension. Individual suspensions of the 6 strains of Salmonella as well as the 6-strain composite suspension were serially diluted in 0.1% bacteriological peptone (Difco) solution and plated on the surface of tryptic soy agar + 0.6% yeast extract (Difco) and incubated for 24 h at 37°C before the number of colonies was counted.
  29. Commercial USDA large grade A shell eggs were purchased from a local grocery store. Five eggs with no cracks or checks were surface-sanitized by immersion in a 70% ethanol solution for 10 s and air-drying on sterile aluminum foil in a laminar flow hood for 30 min. The evening prior to the experiment, eggs were aseptically opened and the yolk and albumen were separated into individual sterile glass beakers with a sterile egg yolk separator. Albumen was surface-plated on tryptic soy agar + 0.6% yeast extract and incubated for 24 h at 37°C to determine mesophilic aerobic background microflora. Albumen (99 mL) was placed into a sterile Waring model CAC32 glass blending jar (Waring Products, Torrington, CT), covered with sterile aluminum foil, and held overnight under refrigeration at 4°C. The blending jar was removed from refrigeration the day of the experiment and allowed to equilibrate to room temperature (21°C) for approximately 1 h and blended with a Waring model LB10G variable speed blender for 1 min on the lowest speed to prevent protein shearing. One milliliter of the 6-strain composite suspension of Salmonella was added to albumen and blended on low speed for 1 min. One milliliter of the inoculated albumen was then serially diluted, spiral-plated on tryptic soy agar + 0.6% yeast extract, and incubated at 37°C for 48 h before enumeration.
  30. Nonselective enrichment, dilution, or neutralizing broths (nutrient broth, universal preenrichment broth, tryptic soy broth, tryptic soy broth without dextrose, lactose broth, Luria-Bertani broth, buffered peptone water, buffered peptone water #2, brain heart infusion broth, and Dey-Engly neutralizing broth; Table 1Go) were solidified either by the addition of 15 g of granulated agar (Difco) per liter before autoclaving, or by the addition of autoclave-sterilized agar at a final concentration of 15 g of granulated agar per liter of broth that had previously been filtered through Whatman no. 2 filter paper (Whatman Inc., Florham Park, NJ) before filter-sterilizing through a Millipore 0.2-µm Steritop Filter (Millipore, Billerica, MA). The chelating compound EDTA was added to tryptic soy agar to determine the effect that binding iron and other metals would have on preventing ROS formation during autoclave-sterilization. Selective media used to recover heat-injured Salmonella were prepared according to the manufacturers’ specifications with the following adjustments: selective enrichment broths (selenite-cystine broth, Rappaport-Vassiliadis broth, and tetrathionate broth; Table 2Go) were solidified by adding 15 g of granulated agar per liter of enrichment broth prior to autoclave sterilization. Bismuth sulfite agar must be carefully prepared to avoid overheating. In this study, bismuth sulfite agar was either prepared according to the manufacturer’s directions, or boiled for 2 min beyond the manufacturer’s specifications, producing a darkened coloration, which is designated as bismuth sulfite agar-dark in Table 2Go. Selective compounds (NaCl, sodium deoxycholate, KMnO4, and calcium hypochlorite) were added to tryptic soy agar (Table 2Go) to determine their level of inhibition on the recovery of injured Salmonella from thermally treated albumen. Media were poured (approximately 12 mL) into sterile Petri dishes. Nonselective media were prepared the day before the experiment and dried for 3 h under a laminar flow hood before plating. Selective media were prepared and held at 21°C for 2 d prior to plating.
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