The Relationship between the Level of Salmonella Enteritidis and the Temperature at Which 

Eggs Have Been Held from the Day of Lay until the Day of Processing 

Final Report - Revised 

Submitted to Dr. David Zeitz 

USDA FSIS OPPED NTS 

By Jinru Chen 

Department of Food Science and Technology 

The University of Georgia 

January 8, 2007 

1 
SUMMARY 

This study was undertaken to determine the relationship between the levels of inoculated 

Salmonella Enteritidis and simulated conditions at which eggs might be held from the day of lay 

until the day of processing. 

Unprocessed chicken eggs (n = 1,920) of different sizes, with 480 of them being laid in winter, 

spring, summer, and fall, respectively were inoculated, in the albumin, with a five-strain mixture 

of Salmonella at 102

 CFU/egg. Eggs not inoculated with Salmonella served as negative controls. 

Both the inoculated and the control eggs were stored at 4, 10, and 22°C (39.2, 50, and 71.6o

F) for 

four weeks. The eggs were sampled twice a week, and at each sampling point, five eggs were 

assayed individually for populations of Salmonella and total aerobic counts. Samples tested 

negative for Salmonella were subjected to enrichment. The experiments were conducted with 

appropriate duplications and replications. None of the control eggs were found to be 

contaminated with Salmonella. Eggs laid in different seasons did not significantly impact the 

survival of the pathogen (P > 0.05). The populations of inoculated Salmonella were not 

significantly different in eggs stored at 4°C versus at 10°C (P > 0.05). However, eggs stored at 

22o

C had an overall Salmonella population that was 3.71 or 3.37 log higher than did the eggs 

stored at 4 and 10oC, respectively (P > 0.05). The overall mean Salmonella population at 22oC 

increased from the initial 2.12 to 3.36 Log CFU/ml after 2 weeks and to 7.84 Log CFU/ml after 3 

weeks into the storage. A sharp increase in the populations of Salmonella occurred after the eggs 

were stored at 22oC for two to two and a half weeks. These results suggest that chicken eggs 

should not be stored at room temperature from the day of lay to the day of processing. 

In a separate experiment, unprocessed chicken eggs (n = 960) of different sizes, with 240 of 

them being laid in winter, spring, summer, and fall, respectively were stored at 4, 10, and 22°C 

for four weeks. The eggs were sampled twice a week, and at each sampling point, the contents 

and shells of five eggs were assayed separately for Salmonella. A total of 14 presumptive 

colonies were isolated and none were confirmed to be Salmonella. Among the 14 isolates, 7 

were Proteus mirabilis, 3 were Enterobacter cloacae, 2 were Pseudomanas, and 2 were 

Escherichia coli. 

2 
INTRODUCTION 

Approximately 67-79 billion eggs are produced in the United States each year (Vogt, 1999; 

USDA, 2000; USDA Economic Resources Service. 2002). Of these, about 20 billion eggs are 

broken, pasteurized, and processed into liquid, dried or frozen egg products and the remaining 

are consumed as whole shell eggs (Vogt, 1999). Georgia is one of the ten largest egg-producing 

states in the country (American Egg Board, 2006) with 11.5 million layers and a total egg 

production of 3 billion. Georgia’s egg industry is very important to the state’s economy, 

accounting for a cash receipt of $368 million (Georgia Egg (Commission, 2006). 

Approximately 1 in every 20,000 eggs produced in the U.S. is contaminated with Salmonella, 

especially with serotype Salmonella Enteritidis (Ebel and Schosser 2002). Contamination of 

eggs by S. Enteritidis is facilitated by the ability of the pathogen to cross the egg shells after the 

eggs are laid (Padron 1990; Chen et al., 1996; Miyamoto et al., 1998; Cox et al., 2000), and 

infect the reproductive systems of healthy hens and contaminate egg contents before egg shells 

are formed (Humphrey 1989; 1991; 1994). However, stringent procedures for cleaning and 

inspecting eggs implemented in the 1970s have made salmonellosis caused by external fecal 

contamination of shell eggs extremely rare (CDC, 1999). The initial level of Salmonella 

contamination in chicken eggs is about 20 cells within 3 weeks of laying (Humphrey et al., 1991; 

Humphrey and Whitehead, 1993). This level of contamination is expected to increase if eggs are 

not appropriately refrigerated after the eggs are laid. Previous research conducted in our 

laboratory, indicated that storage temperature had a significant influence on the quality and 

safety of eggs (Chen et al., 2005). When eggs were stored at 4o

C, their albumen had 

significantly less volume and less weight loss, as well as lower pH, which are indicators of 

slower egg aging process, in comparison to the egg albumen from storage at 10o

 and 22oC. 

Additionally, the vitelline membranes of refrigerated eggs were relatively stronger and required 

greater levels of energy to be ruptured. Salmonella flourished in eggs stored at 22oC, even in the 

eggs with an initial level of contamination of 102 CFU/egg. Storage at 4o and 10oC prevented 

Salmonella cells from growing in the eggs that had an initial inoculation level of 102

 or 104 

CFU/egg. When the initial level of inoculation reached 106 CFU/egg however, the antimicrobial 

agents in the albumen and refrigeration storage were no longer capable of inhibiting the growth 

of Salmonella, and an outgrowth of the pathogen subsequently occurred. 

Salmonella is the second most common cause of human enteric infections and affects 

approximately 1.4 million people each year in the United States with 500 ultimately resulting in 

death. The medical expenses, productivity losses, and number of deaths associated with illness 

caused by salmonellosis are estimated to be in the millions of dollars. In this study, we 

determined the relationship between the level of Salmonella, particularly S. Enteritidis, and the 

temperature at which eggs might be held. The project provides crucial information needed for 

implementation of the HACCP program in small and very small egg farms as well as egg 

processing facilities. It will help egg producers realize the importance of keeping eggs 

refrigerated, and provide regulatory agencies with baselines of Salmonella contamination in 

refrigerated as well as temperature-abused eggs before they enter egg processing facilities. 

3 
 

 

 

Part I: The Relationship between the Level of Salmonella Enteritidis and the Temperature 

at Which Eggs Are Held 

MATERIALS AND METHODS 

Eggs 

Unprocessed chicken eggs (n = 1,920) of different sizes, with 480 of them being laid in January 

(winter season), March (spring season), July (summer season), and September (fall season), 

respectively, were obtained from a commercial processor in Georgia one day before the 

experiments. The eggs were transported under refrigerated conditions from the processor’s farm 

to our laboratory in Griffin GA, and were stored at 4°C until use. Among the 480 eggs used in 

each season, 240 were used in the original experiment, with the remaining 240 in the replicate 

experiment. 

Bacterial Strains and Growth Conditions 

Five strains of Salmonella Enteritidis representing different phage types were used in the study. 

The Salmonella strains were retrieved from frozen stocks and cultivated on tryptic soy agar 

(TSA; Difco, Becton Dickinson, Sparks, MD) and bismuth sulfite agar (BSA; Difco, Becton 

Dickinson, Sparks, MD) at 37°C for 24 h. A single colony of each strain was aseptically 

transferred to tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD) and incubated at 

37°C for 16 h. A cocktail of a five-strain mixture was made by pooling equal aliquots of the five 

cultures. The mix was then appropriately diluted with buffered peptone water (BPW) to a 

concentration of 104

 CFU/ml. 

Inoculation and Storage 

The chicken eggs described above were rinsed with deionized water, surface sterilized with 70% 

ethanol, and wiped with sterile paper towels. Holes on the egg shell were punched using sterile 

sharp-tip forceps. Fifty microliters of the Salmonella mixture at the concentration of 104 

CFU/ml was inoculated into the egg albumen by inserting an 18 gauge needle on a 1 ml syringe 

at the 2 o’clock position in a path approximately parallel to the shell (Schoeni et al., 1995). The 

inoculated eggs were then stored at 4, 10, and 22°C for the period of four weeks to mimic the 

possible conditions under which eggs are held from the day of lay until the day of processing. 

Salmonella Enumeration 

The eggs were sampled twice a week and at each sampling point five eggs were taken from the 

storage and assayed individually for the population of Salmonella Enteritidis and total aerobic 

counts. The experiments have appropriate duplications and replications. To ensure there was no 

prior contamination, negative control eggs were sampled simultaneously with the inoculated 

eggs at each sampling point. Eggs that were cracked during transportation and handling were 

discarded. On each sampling day, eggs were withdrawn from the storage, swabbed with 70% 

ethanol, and cracked with a sterile spatula in a sterile plastic bag. The contents of the eggs were 

homogenized using a stomacher (Seward, UK) for 2 min, and serially diluted in BPW. 

4 
 

 

Appropriate dilutions were plated in duplicate on BSA for the enumeration of Salmonella and on 

TSA for total aerobic counts. The inoculated plates were incubated at 37°C for 24 h and colonies 

appearing on the plates were counted using an automatic colony counter. 

Enrichment 

The samples that tested negative on the BSA plates were subjected to the enrichment procedures 

for Salmonella. Twenty-five ml of the egg contents were transferred in to 225 ml of BPW, and 

the samples were incubated for 24 h at 37°C. One milliliter of the sample was then transferred 

into 9 ml of Rappaport Vassiliadis broth (Difco, Becton Dickinson), and incubated at 41.5°C for 

24 h. A loop full of the enriched cultures was then streaked on BSA plates, and appearing 

colonies were confirmed by biochemical and agglutination tests. 

Statistical Analysis 

Randomized complete block design was used in this study with three different storage 

temperatures (4, 10, and 22°C) and five different storage times (0, 1, 2, 3 and 4 weeks). Data 

was analyzed with the general linear model procedure using Statistical Analysis Software (SAS 

9.1, SAS Institute, Inc., Cary, N.C.) at a 95% confidence level. 

RESULTS 

None of the control eggs were found to be contaminated with S. Enteritidis. The populations of 

total aerobes and the inoculated Salmonella were not significantly different in eggs that were laid 

in different seasons (P > 0.05; Table 1). Figure 1 shows the influence of storage temperature and 

storage time on the fate of artificially inoculated Salmonella in eggs laid in January (Figure 1A), 

March (Figure 1B), July (Figure 1C), and September (Figure 1D), respectively, whereas the 

influence of storage conditions on the population of total aerobes is shown in Figure 2. The 

populations of Salmonella decreased slightly during the four week storage period at 4°C. Only 

in 2 out of the 80 eggs laid in January and stored at 4o

C, did the populations of Salmonella fall 

below the detection levels (1 CFU/ml) however, these 2 eggs were later confirmed positive for 

Salmonella after the enrichment. At 10°C there was no significant increase or decrease in the 

numbers of the Salmonella cells in artificially contaminated eggs. Only 1 out of the 80 eggs laid 

in January and stored at 10o

C tested negative for Salmonella by direct plating. Viable 

Salmonella cells were however, recovered from the eggs after the enrichment procedure. The 

populations of inoculated Salmonella were not significantly different in eggs stored at 4o

C versus 

10o

C (P > 0.05; Table 1). However, eggs stored at 22oC had significantly higher populations of 

Salmonella. At this temperature, the populations of Salmonella in the eggs increased from the 

initial 2.12 Log10 CFU/ml to 3.36 Log10 CFU/ml after 2 weeks and 7.84 Log10 CFU/ml after 3 

weeks into the storage. A sharp increase in the populations of Salmonella occurred after the eggs 

were stored at 22o

C for two to two and a half weeks. A sharp increase in the populations of 

Salmonella and total aerobes occurred after the eggs were stored at 22°C for two or two and a 

half weeks. These results suggest that chicken eggs should not be stored at this temperature from 

the day of lay to the day of processing. 

5 
 

 

 

 

Table 1. Overall mean populations of Salmonella and total aerobic bacteria and results of 

statistical analysis 

Microbiological Media 

TSA BSA 

Season 

Winter 3.49a 3.34a 

Summer 3.29a 3.29a 

Spring 3.02a 2.95a 

Fall 2.89a 2.70a 

Storage Temperature (o

C) 

22 5.40a 5.43a 

10 2.28b 2.06b 

4 1.84b 1.72b 

Storage Time 

3W2 4.63a 4.24a 

3W1 4.26ab 3.96a 

2W2 3.48bc .73ab 

2W1 2.89cd 2.88bc 

1W1 2.68cd 2.60c 

0W2 2.67cd 2.57c 

1W2 2.62cd 2.47c 

0W1 2.15d 2.12c 

*: Means followed by the same letters, in the same column and within the category of sampling 

seasons, storage temperatures, or lengths of storage time, are not significantly different. 

6 
-

-

,------------------

1----,, 

1----r, , , 

-' 

. 

- -

-

A

 B 

Winter Season 

02468

1012

0W1 0W2 1W1 1W2 2W1 2W2 3W1 3W2

Sampling Time 

Salmonella

(Log10 CFU/ml) 

Spring Season 

02468

1012

0W1 0W2 1W1 1W2 2W1 2W2 

Sampling Time 

Salmonella

(Log10 CFU/ml) 

C

 D 

Summer Season 

02468

1012

0W1 0W2 1W1 1W2 2W1 2W2 3W1 3W2

Sampling Time 

Salmonella 

(Log10 CFU/ml) 

Fall Season 

02468

1012

0W1 0W2 1W1 1W2 2W1 2W2 3W

Samping Time 

Salmonella

(Log10CFU/ml) 

Figure 1. Populations of Salmonella Enteritidis in artificially inoculated chicken eggs stored for four weeks at 4

22°C (▲), respectively. A: Data from the winter trial (n = 480), B: Data from the spring trial (n = 480), C: Data 

(n = 480), and D: Data from the fall trial (n = 480). Sampling times included 0 - 3 weeks (0W - 3W) with two s

(sampling 1 - 2) 
- -

- - f' 

- L_n1c---__........,,.... ...... """"'l!""'"="".., - """=~-F = =oll""

A

 B 

12

Winter Season 

12

Spring Season 

10 10

obes

CFU/ml) 8 8 obes

CFU/ml) 

Aer 6 6 AerTotal

(Log10 42

0

420

Total

(Log10

0W1 0W2 1W1 1W2 2W1 2W2 3W1 3W2 0W1 0W2 1W1 1W2 2W1 2W2 

Sampling Time Sampling Time 

C

 D 

Summer Season Fall Season 

12 1210 10

obes

CFU/ml) 8 8 obes 

CFU/ml) 

Aer 6 6 AerTotall

(Log10 42

0

420

Total 

(Log10

0W1 0W2 1W1 1W2 2W1 2W2 3W1 3W2 OW1 OW2 1W1 1W2 2W1 2W2 

Sampling Time Sampling Time 

Figure 2. Populations of total aerobes in artificially inoculated chicken eggs stored for four weeks at 4°C (♦), 

(▲), respectively. A: Data from the winter trial (n = 480); B: Data from the spring trial (n = 480); C: Data from 

480); and D: Data from the fall trial (n = 480). Sampling times included 0 - 3 weeks (0W - 3W) with two sam

(sampling 1-2) 
Part II: Isolation of Salmonella from Chicken Eggs Stored at Different Temperatures 

TASK SUMMARY 

A total of 960 fresh chicken eggs were sampled for Salmonella over one year period, with 240 

eggs being sampled in January, April, July, and September, respectively. The contents as well as 

the shells of all eggs were separated, sampled, and enriched for Salmonella. 

MATERIALS AND METHODS 

Source of Eggs 

During each of the four seasons, a total of 240 freshly laid, unprocessed chicken eggs were 

obtained, on 2 separate occasions, from a commercial egg processor in Georgia. The initial trial 

of the experiment was conducted with the first group of eggs (n = 120), then repeated with the 

second group of eggs (n = 120). Both groups of eggs were transported to our laboratory under 

refrigerated conditions one day prior to the experiments, and stored at 4, 10, and 22o

C, 

respectively with 40 eggs at each temperature in each trial. 

Egg Sampling 

In each trial, egg sampling was performed twice per week during a four week-storage period at 4, 

10, and 22oC, with five eggs being withdrawn from each storage temperature at each sampling 

point. On the day of sampling, each egg was cleaned with running tap water, disinfected with 

70% (v/v) of ethyl alcohol, and dried with sterile paper towels. After cleaning and disinfecting 

the surface, each egg was aseptically cracked open with a sterile metal scoop. The egg was 

separated into shell and liquid egg, and sampled separately. The liquid egg contents were placed 

in a sterile Whirl-Pak bag, and homogenized in a Stomacher Lab Blender (Seward Lab System, 

UK) at normal speed for 2 min. 

Shell Sampling 

The shells of the eggs were transferred to a different sterile Whirl-Pak bag containing 25 ml of 

lactose broth. The shells and egg membranes were gently crushed with a pair of gloved hands 

for approximately 30 seconds (Berrang et al., 1991; Chang 2000). 

Direct Plating 

One ml of each homogenized liquid egg sample and shell-rinsing broth was inoculated separately 

onto bismuth sulfate agar (BSA) plates. The inoculated plates were incubated at 37o

C for 24 h. 

Typical colonies, if any, were selected from the BSA plate and confirmed by biochemical and 

serological tests. 

Salmonella Enrichment 

9 
 

 

After homogenization, 25 ml of liquid eggs or shell-rinsing broth were transferred to a sterile bag 

which contained 225 ml of lactose broth. The samples were allowed to stand at room 

temperature for one hour. The pH of the pre-enrichment mixtures was adjusted to 6.8 ± 0.2 with 

0.1 N HCl or 0.1 N NaOH and 2.25 ml of Triton X-100 (Fisher Scientific, Fair Lawn, NJ) was 

then added. The pre-enrichments were conducted at 37oC for 24 h, and following incubation one 

ml of the pre-enriched cultures was transferred to 10 ml of tetrathionate (TT) broth and 0.1 ml of 

the pre-enriched cultures was transferred to 10 ml of Rappaport-Vassiliadis (RV) broth. The 

inoculated broth was incubated at 42oC for 24 h after which, one ml of each broth culture was 

streaked separately onto BSA plates which were incubated at 37o

C for 24 h. Presumptive 

Salmonella colonies were selected and grown on MacConkey agar at 37o

C for 24 h. Selected 

colorless colonies on MacConkey agar were then inoculated into triple sugar iron agar (TSI) and 

lysine iron agar (LIA) for biochemical confirmation. Isolates from presumptive positive TSI 

slants were confirmed using agglutination assays. All dehydrated media used in the study were 

obtained from Difco Laboratories. 

RESULTS 

Out of the 960 liquid egg samples and 960 egg shells sampled in the project a total of 14 

presumptive Salmonella colonies were recovered from 4 eggs and 6 samples (Table 2). 

However, none of the colonies were confirmed to be Salmonella. Among the 14 isolates, 7 were 

Proteus mirabilis, 3 were Enterobacter cloacae, 2 were Pseudomanas spp., and 2 were 

Escherichia coli. 

PROJECT DELIVERABLES 

The results of the research will be posted on the website of the Food Product Innovation and 

Commercialization Center at The University of Georgia, and perhaps the website of American 

Egg Board and Egg Nutrition Center. An abstract has been submitted to the Institute of Food 

Technologist (IFT) and, upon acceptance, a poster presentation will be made at the annual 

meeting of IFT in July 2007. After which a manuscript will be submitted to the Journal of Food 

Protection for publication in 2007-2008. 

REFERENCES 

American Egg Board. 2006. Egg industry fact sheet – April (2006.) 

http://www.aeb.org/Industry/Facts/FactsSheet.htm. 

Berrang, M. E., N. A. Cox, J. S. Bailey, and L. C. Blankenship. 1991. Methods for inoculation 

and recovery of Salmonella from chicken eggs. Poult. Sci. 70:2267-70. 

CDC. 1999. Prevalence of Salmonella spp. and Escherichia coli on U.S. livestock operations. 

Chang, Y. H. 2000. Prevalence of Salmonella spp. in poultry broilers and shell eggs in Korea. J. 

Food Prot. 63:655-8. 

10 
Chen, J., R. Clarke, and M.W. Griffiths. 1996. Penetration of Salmonella enteritidis through 

whole-shell eggs and survival of the bacteria in whole-liquid egg during refrigeration storage. J. 

Food Prot. 9:915-921. 

Chen, J., H. S. Thesmar, and W. L. Kerr. 2005. The outgrowth of salmonellae and the physical 

property of albumin and vitelline membrane as influenced by egg storage conditions. J. Food 

Prot. 68:2553-2558. 

Cox, N. A., J. S. Bailey, R. J. Buhr, and M. E. Berrang. 2000. Bactericidal treatment of hatching 

eggs. IV. Hydrogen peroxide applied with vacuum and a surfactant to eliminate Salmonella from 

hatching eggs. J. Appl. Poult. Res. 9:530-534. 

Ebel, E. and W. Schosser. 2002. Estimating the annual fraction of eggs contaminated with 

Salmonella enteritidis in the United States. Int. J. Food Microbiol. 61:51-62. 

Georgia Egg Commission. 2006. Governor Purdue proclaims egg month. 

http://www.georgiaeggs.org/pages/news.html. 

Humphrey, T. J., J. G. Cruickshank, B. Rowe, and S. Hopper. 1989. Salmonella enteritidis phage 

type 4 from the contents of intact eggs: a study involving naturally infected hens. Epidemiol. 

Infect. 103:415-423. 

Humphrey, T. J., A. Whitehead, A. H. L. Gawler, A. Henley, and B. Rowe. 1991. Numbers of 

Salmonella enteritidis in the contents of naturally contaminated hens’ eggs. Epidemiol. Infect. 

106:489-496. 

Humphrey, T. J., and A. Whitehead. 1993. Egg age and the growth of Salmonella enteritidis PT4 

in egg contents. Epidemiol. Infect. 11:209-219. 

Humphrey, T. J., K. W. Martin, and A. Whitehead. 1994. Contamination of hands and work 

surfaces with Salmonella enteritidis PT4 during the preparation of egg dishes. Epidemiol. Infect. 

106:489-496. 

Miyamoto, T., K. Sasai, E. Baba, T. Horie, and T. Fukata. 1998. Changes in microflora of the 

cloaca and oviduct of hens after intracloacal or intravaginal inoculated with Salmonella 

enteriridis. Avian Dis. 42:536-544. 

Padron, N. A. 1990. Salmonella typhimurium penetration through the eggshell of hatching eggs. 

Avian Dis. 34:263-465. 

Schoeni, L. J., K. A. Glass, J. L. Mcdermott, A. C. L. Wong. 1995. Growth and penetration of 

Salmonella heidelberg and Salmonella typhimurium in eggs. Int. J. Food Microbiol. 24:385-

386. 

USDA. 2000. Egg production and distribution. http://www.aeb.org/eii/facts/us-prod.html. 

11 
USDA Economic Resources Service. 2002. Brief room: poultry and eggs. 

http://www.ers.usda.gov/Briefing/Poultry/Data/data.htm. 

Vogt, D. U. 1999. Congressional research service report for Congress, Federal regulatory 

structure for egg safety: fact sheet. http://www.cnie.org/NLE/ag-90.html. 

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