Reduction of Escherichia coli O157:H7 and Salmonella spp. using 

Dry Chilling in small processing plant environments 

Tittor, A.W., M.G. Tittor, M.B. Brashears, J.C. Brooks and M.F. Miller 

Texas Tech University Department of Animal and Food Sciences 

Box 42141, Lubbock, Texas 79409 

Introduction 

Small processors are continually searching for avenues to meet HACCP requirements. 

Due to the limitation of space and resources, additional Critical Control Points are welcomed to 

aid in the combat against foodborne pathogens and adulterants. Preliminary data from Texas 

Tech University show dry chilling can reduce the pathogen load on beef surfaces (Woerner et 

al. 2005). Confirmation of the original determination would create an opportunity for Texas 

Tech to aid small processors in providing a CCP for small plants and the necessary 

documentation of its efficacy. Furthermore, most small producers use dry aging up to 28 d for 

tenderness purposes. The objective of the study was determine and quantify the reduction of 

growth of E. coli O157:H7 and Salmonella spp on beef tissue following two chilling methods 

(spray chilling and dry chilling) used by small processing plants, as well as determining the use 

of dry chilling as a CCP. 

Materials and Methods 

Preparation of Pathogen Cocktails. Three strains of E. coli O157:H7 (966, 922, and 944), 

and three strains of Salmonella spp. , (Typhimurium, Hydelburg and Enteritidis) all isolated 

from beef animals or beef products were selected for use in the study. These cultures were 

obtained from the stock culture collection at Texas Tech University. All strains were 

maintained as frozen (-80°C) stocks in TSB with 10% (vol/vol) sterile glycerol as a cryoprotectant. The three strains of E. coli and Salmonella spp. were combined into a cocktail and 

concentrated into frozen cultures as described by Brashears et al. (1999). A frozen stock 

culture of each strain was thawed to room temperature (25-27° C), placed into 200 ml of 

Tryptic Soy Broth and incubated at 37° C for 24 hrs. The cells were separated from the broth 

by centrifugation (4,000 3 g for 20 min at 18oC), and each pellet was re-suspended in 10 ml of 

a 10% solution of sterile glycerol as a cryo-protectant to obtain concentrations of approximately 

109

 cfu/ml. Re-suspended solutions of each strain were mixed in equal volumetric parts to 

obtain a cocktail of three strains for each pathogen. The cocktail suspensions were transferred 

into 2 ml cryogenic vials, quickly frozen in liquid nitrogen, and stored at -80°C. 

Sample Preparation and Inoculation. Hot (25-30° C) adipose tissue, removed from the area 

covering the deep pectoral (brisket), and/or hot lean tissue (25-30° C), Cutaneous omobrachialis (shoulder rose), were removed from carcasses following hide removal, but prior to 

application of any post-mortem antimicrobial washes from a commercial slaughtering facility. 

The tissue samples were transported to Texas Tech University Pathogen Processing Lab 

using unsealed vacuum bags in an ice chest to maintain temperature (25 - 30° C), until 

inoculation (< 3 hrs). Each sample was laid horizontally, aseptically cut with a sterile knife 

blade into approximately 15 cm x 10 cm rectangular portion. All samples were dipped into one 

of two inoculums, three strain E. coli cocktail or three strain Salmonella spp. cocktail from 

frozen concentrate, at a concentration of 107

 cfu/ ml. Following inoculation, each sample was 
 

 

randomly assigned to a treatment and held at room temperature (25°C to 28°C) for 20 min to 

allow for attachment of pathogens. Samples were suspended using sterile fishing hooks and 

line for application of treatments in a refrigerated room (3° C, with 4 defrost time periods of 20 

min) for up to 28 d. 

Definition of Treatments. 

Spray Chill/Wet Ageing. A custom spray chilling system was developed to mimic industry 

application. The system was composed of 36 nozzles with a flow rate of 500 ml/ min with a 

circular pattern. All samples were suspended 30 cm from the nozzles. Suspended tissue 

samples were sprayed continuously for 15 min and then sprayed for 1 min spray cycle every 

17 min for the remainder of the 17 hours. After 48 hrs, all remaining samples were packaged 

in vacuum bags and randomly assigned to an end-point sampling time. Samples were 

excised for enumeration/ detection at the following time periods 0 d, 24 h, 36 h, 48 h, 7 d, 14 d, 

21 d and 28 d. 

Dry Chill/Dry ageing. All samples were suspended in a cold room (3° C, with 4 defrost time 

periods of 20 min) with an air velocity of 0.0 m/ s to 0.25 m/ s with a relative humidity of 80 %. 

Samples were randomly assigned to end point time periods for enumeration/ detection at 0 d, 

24 h, 36 h, 48 h, 7 d, 14 d, 21 d and 28 d. 

Excision of Samples and Enumeration of Pathogens. 

Surface tissue samples measuring 5 cm by 5 cm or 25cm2

 were aseptically removed 

using sterile scalpels and forceps. Samples were placed into sterile stomacher bags, and 25 

ml of buffered peptone water was added. Samples were then stomached at 2500 rpms for 2 

m. Samples inoculated with Salmonella spp. were plated on XLD agar overlaid with TSA using the 

thin-layer agar method to allow for recovery of injured cells (Kang and Fung 2000). Samples containing 

E. coli O157:H7 were plated onto MacConkey Agar with Sorbitol overlaid with TSA using the thin-layer 

agar method as well. Samples were plated using a spiral plating system and counted using a Q count 

automated counting system. All plates were incubated for 18-24 hrs at 37°C prior to counting. In the 

instance no growth was detected, samples then were subjected to the following detection processes. 

Detection. Samples inoculated with E. coli O157 were enriched with buffered peptone water for 24 h at 

37°C. Following enrichment, 1 ml of enrichment was pipetted into 9 ml of GN broth and incubated for 

24 h at 37°C. Then streaked onto MacConkey Agar with Sorbitol, were typical colonies were to be 

observed. 

Samples inoculated with Salmonella spp. were enriched with buffered peptone water for 24 h at 37°C. 

Following enrichment, 1 ml of enrichment was pipetted into 9 ml of RV broth and incubated for 24 h at 

37°C. Then streaked onto XLD selective media, were typical colonies were to be observed. 

Statistical Design and Analysis 

All counts were transformed into log CFU/ cm2 for statistical analyzation. Least square means 

were calculated using the analysis of variance in the general linear model of SAS. Both pathogens 

were analyzed separately and blocked by tissue (lean or fat). Day and treatment were analyzed 

separately as well as interactively. 

Results 

• No recoverable E. coli O157:H7 or Salmonella were found on fat or lean tissue samples of uninoculated (control) samples. 

• Higher numbers of bacteria were recovered from lean tissue samples compared to fat tissue 

samples for both Salmonella and E. coli inoculums (P<0.05). 
 

---------------------------·---------·----,---------·--------

o This is caused by the difference in the higher amount of available water from lean tissue 

samples compared to fat tissue samples. 

Table 1: LS Mean comparison of recovery of E. coli O157:H7 for Lean and Fat Tissues Samples Inoculated E. 

coli O157:H7 and Subjected to either Dry or Spray/Wet Chilling Methods over 28 d, reported in Log of cfu/cm2

. 

# of Tissue Treatment 0 d 24 h 36 h 48 h 7 d 14d 21 d 28 d samples SE 

Lean 

Dry 6.05ax 5.14bx 4.96bx 4.43bcx 3.66cdx 2.98dex 2.89ex 1.10fx 24 .309 

Spray/Wet 6.01ax 4.45bxy 4.49bxy 4.23bxy 4.30bxy 4.07by 3.74bx 3.67by 24 .309 

Dry 5.53ax 4.31bxy 4.24byz 3.85by 2.08cy 2.12cz 0.84dy 0.95dx 24 .309 

Fat 

Spray/Wet 5.75ax 3.61by 3.63bz 3.81by 3.37bz 2.94bz 3.05bx 3.66by 24 .309 

abcdef LS Means with differing superscripts in a row are significantly different (P< 0.05) 

xyz LS Means with differing superscripts in a column are significantly different (P< 0.05) 

• For Lean tissue samples, a difference in treatments did not exists until 14 and 28 d of treatments with 

Spray/wet chilling having significantly more E. coli O157:H7 than recovered from samples from Dry 

Chilling. 

• For Lean tissue samples, as Dry Chilling Storage time increased amount of pathogen recovered from 

tissue decreased with significant reductions from 0d to 24 h, 24 h to 7d, 7 d to 21 d and 21 d to 28 d. 

However, Spray/wet Chilling only had a significant reduction from 0d to 24 h. All other time points for 

Spray/wet Chilling were not significantly different. 

o A constant decrease of pathogens from Dry Chilling is caused by continued desiccation of the 

exterior of the samples. However, an initial decrease was seen in Spray/wet Chilling from a 

washing effect of the water. A numerical decrease in recovery of pathogens over time was seen 

for Spray/wet Chilling, however this decrease was not statistically significant from 24 h to 28 d. 

• For Fat tissue samples at 7d, 21 d and 28 d significantly less E. coli was recovered from samples 

subjected to Dry Chilling than Spray/wet chilling. 

• Fat tissue samples subjected to Dry chilling had significant decreases in recovery as storage time 

increased with 0 d being significantly different from 24 h, 24 h significantly higher in recovery of 

pathogens than 7 d and a significant decrease from 7 d to 21 d. 

Table 2: LS Mean comparison of recovery of Salmonella spp. for Lean and Fat Tissues Samples Inoculated 

Salmonella spp. and Subjected to either Dry or Spray/Wet Chilling Methods over 28 d, reported in Log of cfu/cm2

. 

# of Tissue Treatment 0 d 24 h 36 h 48 h 7 d 14d 21 d 28 d SE samples 

Dry 6.08ax 5.13bx 4.82bx 4.62bx 3.76cx 2.92dy 1.93ey 1.58ey 24 .246 

Lean 

Spray/Wet 6.04 ax 4.95bx 4.58bcx 4.53cx 4.06cdx 3.62dx 3.42dx 3.76dx 24 .246 

Dry 5.71 ax 4.53bx 4.43bx 3.94by 2.94cy 2.29cdy 1.72dey 0.92ey 24 .246 

Fat 

Spray/Wet 5.78 ax 4.14bx 4.16bx 3.30cy 3.03cy 2.68cy 3.26cx 3.11cx 24 .246 

abcdef LS Means with differing superscripts in a row are significantly different (P< 0.05) 

xyz LS Means with differing superscripts in a column are significantly different (P< 0.05) 

• Lean samples inoculated with Salmonella spp. subjected to Dry Chilling over a 28 showed significant 

reductions in pathogens. A significant reduction was shown with a decrease from 0 d to 24h, a significant 

reduction in recovery of Salmonella was also seen from 24 h to 7 d of Dry Chilling. Furthermore, a 

significant decrease was observed from 7 d to 14 d and 14 d to 21 d. 

• For lean samples inoculated with Salmonella subjected to Spray/wet Chilling, a significant decrease was 

observed in recovery of Salmonella from 0 d to 24 h. 

o This reduction is a washing effect of Spray chilling. 

• A significant decrease was seen from 24 h to 48 h of Spray/wet Chilling of lean samples. As well as 48 h 

to 14 d. 
 

 

o Decreases from 24h to 48 h can be explained by the drying out of the samples surfaces. 

• Treatment differences were for lean tissues inoculated with Salmonella were observed at 14 d and 

continued throughout 28 d with Dry Chilled samples having significantly less recovery of Salmonella than 

Spray/wet Chilled samples. 

• Fat samples inoculated with Salmonella subjected to Dry Chilling over 28 d period were observed to 

significantly decrease as time increased. A significant reduction was observed for Dry Chilling samples 

from 0 d to 24 h, 24 h to 7 d, 7 d to 21 d and 21d to 28 d. 

• Fat samples inoculated with Salmonella in which were Spray/wet Chilled showed a significant decrease in 

the quantity of Salmonella recovered from 0d to 24 h. Furthermore a significant decrease was observed 

from 24 h to 48 h, with the remaining time period not being significantly different from the 48 h 

quantification. 

Discussion 

A decrease in recovery was seen across both pathogens, as well as both treatments. However, 

Dry Chilling had the most constant and significant reduction in pathogenic bacteria across the 28 d 

sampling period. Spray/wet Chilling was observed to have an initial drop in recovery of pathogenic 

bacteria, being attributed to a washing effect of the water during the first 17 h. However, Salmonella 

samples continued to slowly decline during the holding period of wet ageing. This would indicate that 

vacuum packing has a bactericidal effect on Salmonella spp. The unexplained rise in recovery of 

pathogenic bacteria, for example 21 d of Salmonella, could have resulted in variation in samples and 

the result of the limited amount of samples that were tested per replication. 

Implications 

Small processors are continually striving to explore cost effective means to reduce pathogenic 

bacteria on beef carcasses. The results from the current study have shown that dry chilling for 21 d is 

an effective intervention in reducing E. coli O157:h7 and Salmonella spp. on fresh beef carcasses 

compared to conventional spray chilling/wet ageing. Currently, small beef or custom beef processors 

allow beef carcass to dry age for 28 d to improve beef palatability. This recommendation is compared 

to Buege and Ingram (2003), in which suggests a 6 day aging of carcasses and critical limits of cooler 

temperature, as well as days of aging. However, recommendation from the above results prove that at 

21 d of aging beef tissue samples can achieve approximately a 4 log reduction of pathogenic bacteria 

by Dry Chilling. Therefore, implicating dry chilling to decrease pathogenic bacteria on the surface of 

beef carcass is effective and economical for small producers. Small processors should validate these 

results in plant setting using indicator organisms. 

Literature Cited 

Buege, D. and S. Ingham. 2003. Small Plant Intervention Treatments to Reduce Bacteria on Beef 

Carcasses at Slaghter. Univ. of Wisconsin Research Update. 

Brashears, M. M. and W. A. Durre. 1999. Inhibitory action of Lactobacillus lactis towards 

Salmonella spp. and E. coli O157:H7 during growth and refrigerated storage. J. Food Prot. 

62:1336-1340 

Kang, D.H. and D. Y. C. Fung. 2000.Application of thin agar layer method for recovery of injured 

Salmonella typhimurium. International Journal of Food Microbiology 54: 127-132 

Woerner, D.R., W.J. Horne, K.R. Underwood, D.M. Cox, Z. L. Vineyard, J.C. Brooks and M.F. Miller. 

2005. Effects of conventional spray chilling versus dry air chilling of beef trim on E. coli O157:H7 and 

Salmonella. 2005 RMC and ICOMST Proceedings.