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

Primary Audience: Quality Assurance Personnel, Researchers, Egg Processing Plant Managers, Regulators

SUMMARY 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 2009 J. Appl. Poult. Res. 18 :297–309 doi: 10.3382/japr.2008-00109

DESCRIPTION OF PROBLEM through the USDA Food Safety and Inspection Service (FSIS) by mandating minimum thermal The USDA National Agricultural Statistics pasteurization requirements for specified egg Service reported that US egg companies pro- products. These standards were based on data duced 2.61 billion pounds of liquid egg in 2007 for the inactivation of Salmonella in liquid egg from 24 billion eggs [1]. The Egg Products products acquired before 1970 and are currently Inspection Act of 1970 regulates egg products being reevaluated in light of recent risk assess-

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. 2 Corresponding author: [email protected] 298 JAPR: Research Report ments, which take into account changes in in- fied M9 minimal salts broth, solidified tryptic dustrial practices such as in-line egg processing soy broth [13], solidified Dey-Engley neutral- and variations in egg product formulations [2, izing medium [14], plate count agar [15], and 3]. Regulations may be amended in accordance tryptic soy agar [16]. Selective plating media with Hazard Analysis and Critical Control Point that have been used to recover Salmonella in and Sanitation Standard Operating Procedure various states of injury by direct plating include compliance guidelines so as to incorporate le- tryptic soy agar + 0.6% yeast extract with 5% thality-based performance standards [4]. sodium chloride [17], bismuth sulfite agar [18], Pasteurized egg products have not been rec- xylose-lysine-tergitol-4 agar [18, 19], solidi- ognized as a source of human illness in the Unit- fied selenite broth, solidified tetrathionate broth ed States since the Egg Products Inspection Act [20], Levine eosin methylene blue agar with 2% of 1970 was issued. However, the USDA FSIS sodium chloride [21], MacConkey sorbitol agar has concluded that current process standards [22], xylose lysine deoxycholate agar, brilliant are not completely effective in eliminating Sal- green agar [10], selenite-cystine agar, Rappa- monella from all egg products, and predictive port-Vassiliadis agar [23], Hektoen enteric agar, models estimate that up to 5,500 people per year Rambach Plus agar, and modified lysine iron may contract salmonellosis from pasteurized agar [24]. egg products [2, 5]. The FSIS reported that dur- Reactive oxygen species (ROS) that are pres- ing routine USDA surveillance, Salmonella was ent in plating media impede the resuscitation of recovered from 0.47% of pasteurized egg prod- injured bacteria. Hydroxyl radicals and other ucts between the years of 1998 and 2003 [4]. ROS produced during autoclave-sterilization of The USDA FSIS risk assessment for Salmonella media, or shortly thereafter, are highly toxic to in shell eggs reported that current minimum bacterial DNA, membrane lipids, and cellular pasteurization standards impart 5.9, 5.5, and 3.3 proteins [25]; thus, various oxygen tension-re- log10 reductions of Salmonella in liquid whole ducing compounds have been added to selective egg, liquid egg yolk, and liquid egg white (albu- and nonselective media to enhance the recovery men), respectively [2]. They estimated that if all of injured bacteria [26]. liquid egg whites in the United States were pas- This study was designed to assess the rela- teurized for a 6-log10 reduction of Salmonella, tive proficiency of 22 selective and 26 nonselec- the annual number of human illnesses from pas- tive plating media in colony development of a teurized egg albumen products could be reduced 6-strain composite of Salmonella from thermally from 2,600 to 270 per year. treated albumen to select media that will maxi- Problems relating to recovering Salmonella mize the recovery of heat-injured Salmonella or from egg products containing yolk have been indicate the level of injury for future egg-pas- reported [6–8], although recovery of Salmonella teurization studies. An investigation of selec- from albumen has proven most difficult [9, 10]. tive compounds (NaCl, KMnO4, EDTA, sodium Innate albumen antimicrobials (e.g., ovotrans- deoxycholate, and calcium hypochlorite) added ferrin, lysozyme, avidin, etc.) [3, 11] may com- to tryptic soy agar was also undertaken to de- pound injury that Salmonella incurs during termine their inhibiting or resuscitating effects pasteurization, complicating bacterial recovery on the Salmonella composite and to assess their and enumeration. Studies that are performed to potential for use in future experiments. assess the presence and levels of Salmonella in pasteurized liquid egg products by direct plat- MATERIALS AND METHODS ing require the use of media with a high level of Bacterial Strains and Preparation sensitivity that will maximize resuscitation and of Cultures for Heat Treatment enumeration of thermally injured bacteria. Non- selective plating media that have been used to Six strains [27] of Salmonella enterica were recover Salmonella in various states of injury by examined for their response to thermal stress in direct plating include brain heart infusion agar liquid egg white and recovery on 26 nonselec- [9, 12], solidified buffered peptone water, solidi- tive and 22 selective media. Cells were grown Gurtler: MEDIA FOR RECOVERING SALMONELLA 299 for 24 h in a nutritive broth and were concen- ionized water, and opened aseptically, and then trated 10-fold [28]. 0.7 mL of the heated albumen was removed with a pipette and added to 6.3 mL of 0.1% peptone Preparation of Egg Albumen solution. Serial dilutions of thermally treated al- and Recovery Media bumen were spiral plated [34] in duplicate on the surface of nonselective or selective agar. All Albumen was prepared [29] and inoculated plates were incubated at 37°C for 48 h and colo- with a 6-strain composite of salmonellae to 7.74 nies were counted [35]. The pH and oxidation- log cells/mL. All media (Tables 1 and 2) were reduction potential (ORP) of each medium and purchased in dehydrated form and prepared ac- albumen sample were measured [36] in dupli- cording to the manufacturers’ specifications [30] cate. unless noted otherwise in the References and Notes. Statistical Analysis Thermal Treatment of Inoculated Albumen Three replicate trials were conducted, and in Thermal-Death-Time Disks duplicate samples were averaged before statistical analysis. Data were analyzed by ANOVA Inoculated albumen was thermally treated by using SAS software version 9.1 [37]. Significant means of aluminum thermal-death-time (TDT) differences (P ≤ 0.05) in the recovery of Salmo- disks, which have been described by Jin et al. nella within selective or nonselective media [31]. Sterile TDT disks were loaded with 0.9 mL were determined by LSD. 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 opti- RESULTS AND DISCUSSION mized in preliminary tests (data not shown) to Heating and Cooling Times effect an approximate 2-log reduction in population and to induce thermally mediated bacterial The heating time needed to increase samples injury to cells in the surviving population. Tem- from 21°C to within 0.5°C of the target temperatures were monitored with type K thermo- perature (53.3°C) was 40 s, and samples were couples and electronically recorded [33]. One held for an additional 3.1 min. The cooling times thermocouple was placed directly in the center needed to lower the temperature from 53.3°C to of the water bath to measure ambient water tem- 15.0 and 0°C were 12 and 55 s, respectively. perature. A second thermocouple was affixed inside a TDT disk through a hole drilled in the Results of pH and ORP Measurements bottom of the disk, sealed with a waterproof ad- hesive, and filled with 0.9 mL of uninoculated After inoculation, the average pH of albumen albumen to monitor the heating time and treat- was 9.0 and the average ORP measurement was ment temperature for each experiment. Heating 114 mV. There were few differences in pH and times began when the internal temperature of the ORP measurements within selective and nonse- TDT disk reached within 0.5°C (52.8°C) of the lective media (Tables 3 and 4). All nonselective target temperature (53.3°C). After the thermal media pH values were between 6.31 and 7.55, treatment, disks were immediately immersed in with ORP readings between 170 and 263 mV, a slush ice bath to arrest the thermal effects on except for presterilization and poststerilization bacterial cells. Dey-Engly neutralizing broth, which had pH values of 8.03 and 8.37 and had ORP readings Plating of Thermally Treated Cells of 80 and 114 mV, respectively. Selective media on Selective and Nonselective Media pH readings were between 6.40 and 7.76, ex- and Measurement of pH and cept for Rappaport-Vassiliadis broth and tryptic Oxidation-Reduction Potential soy agar + 0.1% KMnO4, which had pH values of 5.34 and 8.37, respectively. Selective media The TDT disks were surface-sanitized by im- ORP values were between 129 and 235, except mersion in 70% ethanol, rinsed with sterile de- for tetrathionate broth and overheated bismuth 300 JAPR: Research Report

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

Additional Step in preparation Abbreviation ingredients added of media at which used for medium Medium Source per liter of water agar was added in this study

Brain heart infusion agar Difco1 —2 — BHIA Brain heart infusion broth Difco 15 g of granulated agar Poststerilization3 BHIB Buffered peptone water Difco 15 g of granulated agar Poststerilization BPW Buffered peptone water #2 Difco 15 g of granulated agar Presterilization4 BPW #2-Pre Buffered peptone water #2 Difco 15 g of granulated agar Poststerilization BPW #2-Post Davis minimal agar Difco — — DMA Dey-Engly neutralizing broth Difco 15 g of granulated agar Presterilization DE-Pre Dey-Engly neutralizing broth Difco 15 g of granulated agar Poststerilization DE-Post Dextrose tryptone agar Sigma5 — — DTA Lactose broth Oxoid6 15 g of granulated agar Presterilization LaB-Pre Lactose broth Oxoid 15 g of granulated agar Poststerilization LaB-Post Levine eosin methylene blue agar Difco — — EMBA Luria-Bertani broth, Miller Difco 15 g of granulated agar Presterilization LuB-Pre Luria-Bertani broth, Miller Difco 15 g of granulated agar Poststerilization LuB-Post Nutrient broth Difco 15 g of granulated agar Presterilization NB-Pre Nutrient broth Difco 15 g of granulated agar Poststerilization NB-Post Plate count agar Company A — — PCA (company A) Plate count agar Company B — — PCA (company B) Plate count agar Company C — — PCA (company C) Tryptic soy agar Difco — — TSA Tryptic soy agar Difco 0.1 g of EDTA — TSA + 0.01% EDTA Tryptic soy broth Difco 15 g of granulated agar Presterilization TSBG-Pre Tryptic soy broth Difco 15 g of granulated agar Poststerilization TSBG-Post Tryptic soy broth without dextrose Difco 15 g of granulated agar Presterilization TSB-Pre Tryptic soy broth without dextrose Difco 15 g of granulated agar Poststerilization TSB-Post Universal preenrichment broth Difco 15 g of granulated agar Presterilization UPB-Pre Universal preenrichment broth Difco 15 g of granulated agar Poststerilization UPB-Post 1Difco (Beckton Dickinson, Sparks, MD). 2A dash (—) indicates not applicable or no ingredients added. 3Filter-sterilized commercial broth was combined with melted agar that had been autoclave-sterilized at 121°C and 15 psi for 15 min. Granulated agar was added at a final concentration of 15 g/L on a weight per volume basis. 4Granulated agar was combined with commercial broth ingredients at a final concentration of 15 g/L on a weight per volume basis during media preparation and before autoclave sterilization at 121°C and 15 psi for 15 min. 5Sigma-Aldrich Corp. (St. Louis, MO). 6Oxoid Inc. (Basingstoke, Hampshire, UK). sulfite agar, with ORP readings of 82 and 104 philic aerobic microorganisms were recovered mV, respectively. Overall, differences in pH and from uninoculated albumen samples. Inoculated ORP values did not appear to be a major factor albumen contained an average of 7.74 log cells/ in the proficiency of media to recover injured mL of Salmonella before heating. cells. All media had ORP and pH values within the ranges reported for supporting the growth of Recovery of Heat-Stressed Salmonella Salmonella [38–40]. from Thermally Treated Albumen on Nonselective Agar Population of Salmonella in Inocula and Albumen Before Heating Mean composite values from direct plating on nonselective agar were analyzed to determine Average populations of inocula were 9.94 significant differences in the numbers of Salmo- log cfu/mL for the 6 individual strains and 9.98 nella recovered on each medium, and are sepa- log cfu/mL for the 6-strain composite. No meso- rated into 6 groupings to simplify discussion Gurtler: MEDIA FOR RECOVERING SALMONELLA 301

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

Additional Abbreviation ingredients added used for medium Medium Source per liter of water in this study

Brilliant green agar Difco1 —2 BGA Brilliant green agar with phosphates Sigma3 — BGAP Brilliant green bile agar Difco — BGBA Bismuth sulfite agar Oxoid4 — BSA Bismuth sulfite agar (overheated) Oxoid — BSA-dark Levine eosin methylene blue agar Difco — EMBA MacConkey sorbitol agar Difco — MSA Rappaport-Vassiliadis broth Oxoid 15 g of granulated agar5 RVA Tetrathionate broth Difco 15 g of granulated agar TTA Selenite-cystine broth Difco 15 g of granulated agar SCA Tryptic soy agar Difco 1 g of calcium hypochlorite TSA + 0.1% calcium hypochlorite Tryptic soy agar Difco 5 g of calcium hypochlorite TSA + 0.5% calcium hypochlorite

Tryptic soy agar Difco 1 g of potassium permanganate TSA + 0.1% KMnO4 Tryptic soy agar Difco 10 g of sodium chloride TSA + 1.5% NaCl Tryptic soy agar Difco 25 g of sodium chloride TSA + 3.0% NaCl Tryptic soy agar Difco 40 g of sodium chloride TSA + 4.5% NaCl Tryptic soy agar Difco 1 g of sodium deoxycholate TSA + 0.1% sodium deoxycholate Tryptic soy agar Difco 2.5 g of sodium deoxycholate TSA + 0.25% sodium deoxycholate Tryptic soy agar Difco 4 g of sodium deoxycholate TSA + 0.4% sodium deoxycholate Violet red bile agar Difco — VRBA Xylose lysine deoxycholate agar Remel6 — XLD Xylose-lysine-tergitol-4 agar Difco — XLT4 1Difco (Beckton Dickinson, Sparks, MD). 2A dash (—) indicates not applicable or no ingredients added. 3Sigma-Aldrich Corp. (St. Louis, MO). 4Oxoid Inc. (Basingstoke, Hampshire, UK). 5Granulated agar was combined with commercial broth ingredients at a final concentration of 15 g/L on a weight per volume basis during media preparation and before autoclave sterilization at 121°C and 15 psi for 15 min. 6Remel Inc. (Lenexa, KS).

(Figure 1). Group I, which included tryptic soy presence of lethal ROS in agar may be related agar + 0.01 EDTA, plate count agar (company to metal-catalyzed hydroxyl production, which A), tryptic soy agar, dextrose tryptone agar, pre- is enhanced after high-temperature sterilization. sterilization nutrient broth, and presterilization Fenton-mediated hydroxyl radical formation oc- universal preenrichment broth, recovered the curs when hydrogen peroxide (generated in mi- greatest numbers of heat-stressed Salmonella, crobiological media during heating) initiates the although only tryptic soy agar + 0.01% EDTA conversion of ferrous iron (Fe2+) to ferric iron recovered significantly more Salmonella than (Fe3+) [42, 43]. Hydroxyl radicals are the most the media in group II, which included postster- reactive oxygen radical and are known to oxi- ilization universal preenrichment broth, post- dize lipids, nucleic acids, and proteins [44]. Ch- sterilization nutrient broth, and presterilization elators can block the generation of hydroxyls by lactose broth. The chelating agent EDTA, at sub- sequestering unbound iron [45]. McFeters and bacteriostatic levels, may provide protection to Singh [46] reported that varying medium com- injured cells by sequestering iron to prevent positions and incubation temperatures as well as ROS generation. Facon and Skura [41] reported the added chelating compounds were all factors that the numbers of Salmonella Enteritidis held in the recovery of injured bacteria. for 4 h at 37°C in tryptic soy broth with 25 mM The numbers of Salmonella recovered on EDTA did not decline when compared with dextrose tryptone agar were equal to those on all holding in tryptic soy broth without EDTA. The media in group I. Dextrose tryptone agar could 302 JAPR: Research Report

Table 3. Oxidation-reduction potential (ORP) and No significant differences in the recovery of pH of nonselective media used to recover a 6-strain Salmonella occurred when granulated agar was composite of Salmonella from egg albumen heated at added to nonselective media before autoclave 53.3°C for 3.1 min sterilization rather than when melted autoclave- Medium1 pH ORP, mV sterilized agar was added to filter-sterilized BHIA 7.35 208 broths (i.e., poststerilization universal preen- BHIB 7.55 226 richment broth, poststerilization nutrient broth, BPW 7.26 236 poststerilization lactose broth, poststerilization BPW #2-Pre 7.22 222 Luria-Bertani broth, poststerilization tryptic soy BPW #2-Post 7.28 238 broth, poststerilization buffered peptone water DMA 7.06 228 DE-Pre 8.03 80 #2, and poststerilization Dey-Engly neutraliz- DE-Post 8.37 114 ing broth) before dispensing into Petri dishes. DTA 6.78 225 Four media in group I (tryptic soy agar + 0.01% LaB-Pre 6.86 237 EDTA, plate count agar [company A], tryptic LaB-Post 6.59 260 soy agar, and dextrose tryptone agar) recovered LuB-Pre 6.86 196 LuB-Post 6.66 263 greater numbers of Salmonella than other media NB-Pre 6.83 248 reported to have been used to recover injured NB-Post 6.79 247 Salmonella (i.e., brain heart infusion agar, buff- PCA (company A) 6.98 231 ered peptone water, presterilization tryptic soy PCA (company B) 7.02 217 broth, presterilization Dey-Engly neutralizing PCA (company C) 6.35 236 TSA 7.46 170 broth, plate count agar [company A], and plate TSA + 0.01% EDTA 7.43 187 count agar [company B]). Augustin and Carli- TSBG-Pre 7.27 211 er [47] reported that culture medium type and TSBG-Post 7.40 205 manufacturer significantly affected the recovery TSB-Pre 7.21 213 of Enterobacteriaceae. Stephens et al. [44] dem- TSB-Post 7.28 216 UPB-Pre 6.31 237 UPB-Post 6.34 239 1For media, see Table 1. 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 be useful in recovering injured Salmonella as 1 well as in differentiating changes in pH because Medium pH ORP, mV it contains bromcresol purple, a pH indicator. So- BGA 6.40 208 lidified universal preenrichment broth, although BGAP 6.72 196 containing sodium pyruvate, which is known to BGBA 6.73 220 enhance the recovery of heat-injured bacteria, BSA 7.40 133 BSA-dark 7.71 104 recovered no greater numbers of Salmonella EMBA 7.07 179 than did plate count agar (company A), tryptic MSA 7.17 180 soy agar, or dextrose tryptone agar, which con- RVA 5.34 222 tained no sodium pyruvate. The use of solidified TTA 7.76 82 enrichment broths (lactose broth, Luria-Bertani SCA 7.11 189 TSA + 0.1% calcium hypochlorite 7.49 231 broth, buffered peptone water, buffered peptone TSA + 0.1% KMnO4 8.37 205 water #2, tryptic soy broth, brain heart infusion TSA + 1.5% NaCl 7.40 235 broth, and nutrient broth) imparted no benefit TSA + 3.0% NaCl 7.37 231 in recovering Salmonella when compared with TSA + 4.5% NaCl 7.35 198 group I media or other commonly used nonse- TSA + 0.1% sodium deoxycholate 7.46 190 TSA + 0.25% sodium deoxycholate 7.46 195 lective recovery media evaluated. Salmonella TSA + 0.4% sodium deoxycholate 7.49 193 recovery differed little (≤0.52 log cfu/mL) VRBA 7.27 178 among varying brands of the nonselective me- XLD 7.57 140 dia tryptic soy agar, plate count agar, dextrose XLT4 7.50 129 tryptone agar, or brain heart infusion agar. 1For media, see Table 2. Gurtler: MEDIA FOR RECOVERING SALMONELLA 303 onstrated a greater than 1,000-fold difference in Recovery of Heat-Stressed Salmonella from heat-stressed Salmonella Typhimurium recov- Thermally Treated Albumen on Selective Agar ered among peptone from 12 sources. Although there have been reports of injured Mean values from selective agar were ana- Salmonella recovering in higher numbers on a lyzed to determine significant differences in minimal medium than on tryptic soy agar, fewer Salmonella recovered on each medium and have cells in this study (4.87 log cfu/mL) were recov- been divided into 5 groupings to simplify dis- ered on Davis minimal agar (group V) than on cussion (Figure 2). Media in group I (brilliant media in groups I to IV (5.29 to 5.94 log cfu/ green agar, Levine eosin methylene blue agar, mL). Mackey and Derrick [20] recovered equiv- brilliant green agar with phosphates, bismuth alent numbers of healthy Salmonella Typhimuri- sulfite agar, and brilliant green bile agar) recov- um LT2 on nutrient agar and solidified M9 mini- ered the greatest number of injured cells among mum salts broth (7.85 cfu/mL), although after commercially available selective media in this heating at 52°C for 90 min in phosphate buffer, study. Although there was no statistical differ- populations of Salmonella recovered were 5.93 ence in the recovery of Salmonella among me- and 5.19 log cfu/mL, respectively. Wilson and dia in group I, only brilliant green agar recov- Davies [48] reported that solidified M9 minimal ered a significantly greater number of injured broth recovered greater numbers of Salmonella Salmonella (5.84 log cfu/mL) than groups II to than tryptic soy agar with 0.5% yeast extract af- V (0.77 to 5.06 log cfu/mL). When raw data for ter cells were heated in phosphate buffer at 52°C Salmonella recovered on nonselective tryptic for up to 30 min. Others have reported similar soy agar were included in the statistical analy- results when heat-injured cells were allowed a sis of Salmonella recovered on selective media, brief recovery period in M9 minimal broth [49]. only the 8 selective media in group I recovered Mackey and Derrick [50] also reported that M9 equivalent numbers of injured Salmonella (5.31 minimal agar was superior to tryptic soy agar to 5.84 log cfu/mL) as tryptic soy agar (log 5.80 with yeast extract for recovering cold-shocked cfu/mL; Figure 1). Salmonella. They hypothesized that the differ- Although not statistically different, fewer ences in recovery could be attributed to lethal injured cells were recovered on overheated bis- peroxide concentrations in tryptic soy agar with muth sulfite agar than on bismuth sulfite agar yeast extract that were measured at 12 to 30 (5.05 and 5.58 log cfu/mL, respectively), dem- μmol/L, whereas no peroxide was detected in onstrating the decreased sensitivity of bismuth M9 agar. Differences in medium composition sulfite agar when overheated. Except for tetra- between M9 minimal agar (which contains dipo- thionate broth (group IV), fewer cells were re- tassium phosphate, monopotassium phosphate, covered on xylose-lysine-tergitol-4 agar than sodium chloride, and ammonium chloride) and on all other commercially produced selective Davis minimal agar (which contains contains media or solidified selective enrichment broths, dipotassium phosphate, monopotassium phos- emphasizing the decreased sensitivity of xylose- phate, dextrose, sodium citrate, magnesium lysine-tergitol-4 in recovering thermally stressed sulfate, and ammonium sulfate) may account salmonellae. Williams et al. [18] recovered few- for the variation in the ability to recover injured er Salmonella on xylose-lysine-tergitol-4 than Salmonella in the present study. Adding nutri- bismuth sulfite agar from apple cider inocu- ents and buffers to a medium has been known lated with salmonellae treated with ozone at 4 to intensify substrate-accelerated bacterial death or 50°C, as well as from Salmonella-inoculated [51–54]. It has been hypothesized that autoxi- orange juice treated with ozone at 50°C. dation of ingredients, such as phosphate buffers There were no significant differences in the and sugars, during autoclaving or shortly there- recovery of injured cells among varying con- after may lead to the formation of hydrogen centrations of sodium deoxycholate added to peroxide and other toxic oxygen species, which tryptic soy agar, although there was a 0.63, 0.88, would inhibit resuscitation of injured bacteria and 1.2 log cfu/mL reduction in the recovery of [20, 44, 50, 55–59]. Salmonella on tryptic soy agar + 0.1, 0.25, and 304 JAPR: Research Report

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. Gurtler: MEDIA FOR RECOVERING SALMONELLA 305

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. 306 JAPR: Research Report

0.4% sodium deoxycholate, respectively, when thin agar layer method has been used to recover compared with the highest level of Salmonella heat-injured Salmonella by plating bacterial sus- recovered on nonselective media (5.94 log cfu/ pensions on tryptic soy agar overlaid with xylo- mL; Figure 1). There was no significant dif- se lysine deoxycholate agar [62]. McCann et al. ference in the numbers of injured Salmonella [63] plated heat-injured Salmonella on tryptic recovered on tryptic soy agar + 0.5% calcium soy agar held at 25°C for 2 h and then overlaid hypochlorite or tryptic soy agar + 4.5% NaCl with xylose lysine deoxycholate agar. The pres- (1.47 and 0.77 log cfu/mL, respectively; group ent study indicates that because 1.81, 2.58, and V) or between tryptic soy agar + 0.1% KMnO4 3.29 log cfu/mL fewer heat-injured Salmonella and Rappaport-Vassiliadis broth (4.34 and 4.07 were recovered on xylose lysine deoxycholate, log cfu/mL, respectively; group III). Addition of xylose-lysine-tergitol-4, and tetrathionate broth, these selective compounds to media may assist respectively, as compared with those recovered in determining the degree of bacterial injury in on brilliant green agar, these media may serve future studies. However, further investigations as good indicators of bacterial injury. These would need to address the sensitivity of these findings may also assist in choosing a selective compounds in recovering healthy, unstressed medium to overlay onto nonselective agar for cells when compared with heat-injured cells. the recovery of heat-injured Salmonella from Rayman et al. [60] reported that tryptic soy agar food samples with a heterogeneous population + 0.15% KMnO4 recovered at least 100 times of bacteria. In selecting an appropriate selec- higher counts of heat-stressed Salmonella Sen- tive medium, one would also need to account ftenberg than tryptic soy agar alone, although for agar specificity, which would largely depend exposure to KMnO4 at higher concentrations is on the population and makeup of background known to be bactericidal [61]. Addition of 0.1% microflora present in a food sample. The addi- KMnO4 to tryptic soy agar in this study, howev- tion of ROS-reducing and other resuscitation- er, did not enhance recovery, and at 0.5%, dete- promoting compounds to selective or nonselec- rioration of media occurred during autoclaving. tive media examined in this study could further Results in the present study indicate that enhance the recovery of heat-injured Salmonella selective and nonselective media vary in their from liquid egg products. ability to support resuscitation and colony formation by Salmonella from thermally treated albumen. Six nonselective media (tryptic soy agar CONCLUSIONS AND APPLICATIONS + 0.01% EDTA, plate count agar [company A], tryptic soy agar, dextrose tryptone agar, prester- 1. The nonselective media tryptic soy agar, ilization nutrient broth, and presterilization uni- plate count agar, dextrose tryptone agar, versal preenrichment broth) supported recovery and brain heart infusion agar all recov- of the greatest, yet equivalent, numbers of Sal- ered maximum and equivalent numbers monella and may assist in choosing a nonselec- of heat-injured Salmonella from egg al- tive recovery medium. This study also provides bumen. information that may be useful in choosing a 2. Selective media that optimized recovery commercially produced selective medium (e.g., of salmonellae from thermally treated al- brilliant green agar, Levine eosin methylene bumen included 3 brilliant green agars, blue agar, brilliant green agar with phosphates, Levine eosin methylene blue agar, and bismuth sulfite agar, and brilliant green bile agar bismuth sulfite agar. recovered the greatest, yet equivalent, numbers 3. Commercially available selective media of cells) to maximize the recovery of injured recovering the fewest cells, which may Salmonella after a period of resuscitation on be used to assess injury of Salmonella a nonselective agar. Various studies have used in pasteurized albumen, were xylose- methods of resuscitating injured cells by incu- lysine-tergitol-4, xylose lysine deoxy- bating on nonselective agar for a specified time, cholate, selenite-cystine, tetrathionate, followed by exposure to a selective medium. A and Rappaport-Vassiliadis agars. Gurtler: MEDIA FOR RECOVERING SALMONELLA 307

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Heat sterilized reducing 76:32–40. sugars and their effects on the thermal resistance of bacteria. J. Bacteriol. 36:369–382. Acknowledgments 56. Baylis, C. L., S. MacPhee, and R. P. Betts. 2000. The author thanks Larry Beuchat (Center for Food Safety, Comparison of methods for the recovery and detection of University of Georgia, Griffin), Tony Jin, and Hyun-Gyun low levels of injured Salmonella in ice cream and milk pow- Yuk (USDA, Eastern Regional Research Center) for review- der. Lett. Appl. Microbiol. 30:320–324. ing this manuscript and providing helpful feedback.