DESCRIPTION OF PROBLEM. Primary Audience: Quality Assurance Personnel, Directors of Research, Microbiologists

2001 Poultry Science Association, Inc. EVALUATION OF THE BIOSYS OPTICAL METHOD FOR RAPIDLY ENUMERATING POPULATIONS OF AEROBIC BACTERIA, COLIFORMS, AND ESCHERICHIA COLI FROM BROILER CHICKEN CARCASSES S. M. RUSSELL Department of Poultry Science, The University of Georgia, Athens, GA 30602-2772 Phone: 706-542-1368 FAX: 706-542-1827 e-mail: srussell@arches.uga.edu Primary Audience: Quality Assurance Personnel, Directors of Research, Microbiologists SUMMARY A study was conducted to determine if a rapid optical method could be used to enumerate total aerobic bacteria, coliforms, and Escherichia coli from broiler chicken carcasses. The BioSys optical method was compared with standard methods for enumerating bacteria. For chicken carcasses, the correlation coefficients for the regression lines comparing aerobic plate counts, coliform counts, and E. coli most probable numbers (MPN) to BioSys [1] optical method measurements were 0.92, 0.92, and 0.91, respectively. Results demonstrated that, using the BioSys method, data were obtained in 2 to 11 h, rather than the 48 h required to conduct aerobic plate counts (APC) or coliform counts or the 8 d required to conduct the MPN procedure. These methods would allow processors to test a product and obtain results prior to shipping the product, avoiding the cost and loss of reputation associated with a recall or food-borne illness outbreak. In addition, the histogram program provided with the BioSys System would allow easy detection of samples that exceed customer or U.S.D.A. specification limits. Key words: BioSys, rapid enumeration, total viable count, coliforms, Escherichia coli, chicken 2001 J. Appl. Poult. Res. 10:141 149 DESCRIPTION OF PROBLEM The Centers for Disease Prevention and Control reported that food-borne disease is one of the most common causes of illness and death among U.S. citizens. Dangerous pathogenic microorganisms associated with foods are estimated to cause between 6 and 81 million illnesses and 9,000 deaths per year throughout the U.S. [2, 3, 4, 5]. To maintain the sanitary quality and safety of poultry products, U.S. companies regularly monitor food contact surfaces and various poultry products for indicator groups of bacteria, such as total aerobes, total coliforms, and Escherichia coli. Aerobic plate counts are usually conducted immediately after the plant has been cleaned and sanitized to determine how efficiently bacterial populations have been eliminated. Sanitation swabs and APC are generally conducted as part of a processing plant s sanitation standard operating procedures as required by the U.S.D.A. Total

142 coliform and E. coli counts are conducted on products to evaluate whether or not the products have been exposed to fecal contamination. The U.S.D.A.-F.S.I.S. [6] reported that nonpathogenic E. coli is the most appropriate microbial indicator of fecal contamination on chicken and beef carcasses, and fecal contamination is a major pathway by which pathogenic bacteria, such as Salmonella, Campylobacter and E. coli O157:H7, gain access to these food products. In addition, E. coli was chosen by the U.S.D.A.-F.S.I.S. to be the indicator of fecal contamination because 1) E. coli enumerations are useful to ensure that processing parameters are in control; 2) analyses are easy and inexpensive to perform; and 3) levels of E. coli can be easily quantified. However, many pathogens are difficult to quantify [7]. Moreover, the U.S.D.A.-F.S.I.S has recently required that all plants operate according to the F.S.I.S. Pathogen Reduction/Hazard Analysis Critical Control Point (HACCP) Regulation, which requires that all poultry and beef slaughter facilities begin enumerating E. coli on carcasses. The level of E. coli that is considered acceptable for poultry products is below 100 colony-forming units (cfu)/ml; a level of 100 to 1,000 cfu/ml is considered marginal. Escherichia coli levels above 1,000 cfu/ml are considered unacceptable [8]. The Pathogen Reduction Regulation requires plants to evaluate one carcass for every 22,000 broiler chickens processed per day [8]. Because an average poultry plant processes approximately 250,000 carcasses per day, 11 carcasses must be analyzed for E. coli each day. Traditional methods for enumerating APC, coliforms, and E. coli are tedious, time-consuming, laborious, and expensive, and the results are not able to be obtained for 2 to 8 d. Because poultry is sold as a fresh, perishable product, by the time microbiological results are obtained, the product has been sold and consumed. To conduct APC using traditional methods, media must be prepared, samples must be diluted by hand, media must be poured (plate count agar) and allowed to solidify, and plates must be incubated for 48 h. After incubation, plates with 25 to 250 cfu are manually counted [9]. This method requires that the technician be skilled in microbiological procedures. The JAPR: Research Report method costs approximately $10 per sample to conduct and is subject to diluting, pipetting, and counting error. The traditional method for enumerating coliforms involves the same procedure as that used for APC, except that violetred-bile agar (VRBA) is used instead of plate count agar, and the agar is overlayed with more VRBA medium once the plates (mixed with the sample) have solidified [10]. For E. coli a much more complex MPN procedure is used. The MPN procedure is performed by inoculating three replicate tubes of lauryl sulfate tryptose (LST) broth with samples diluted to 10 1, 10 2, and 10 3. Within each LST tube, an inverted Durham tube is inserted during media preparation, and then the tubes are incubated at 35 C for 48 h. All LST tubes that are positive for gas production in the inverted Durham tubes are cultured in E. coli broth (with Durham tubes) by incubating the E. coli tubes at 45.5 C for 48 h. Escherichia coli tubes that are positive for gas production are streaked on Levine eosin methylene blue agar plates and incubated at 35 C for 24 h. Dark-centered colonies with a metallic sheen are then selected and transferred onto plate count agar slants and incubated at 35 C for 24 h. Gram stains and indole, methyl red, Voges-Proskauer, and citrate (IMViC) tests are then performed. The LST tubes are inoculated from the plate count agar slants and incubated at 35 C for 48 h. The MPN of E. coli is determined using an MPN chart, based on a Gram-negative stain, IMViC test patterns; and gas production in the LST tubes. This process takes a total of approximately 8 d to perform [10]. In 1997, Shelef and Firstenberg-Eden [11] described a method for detecting metabolic changes that occur as bacteria multiply in growth medium. Optical changes were monitored in a semi-fluid zone that separates the liquid medium containing the sample from the reading area. Nutrient broth with a ph dye indicator was used to assess the presence or absence of certain species of bacteria [11]. Additionally, Shelef et al. [12] used the optical method for estimating microbial contamination of prewrapped fresh ground beef. The authors found that this method can provide a good estimate of the microbial quality of fresh meat. The purpose of this research was to determine if a rapid

RUSSELL: RAPID ENUMERATION OF BACTERIA 143 optical method could be used to enumerate total aerobic bacteria, coliforms, and E. coli from broiler chicken carcasses. MATERIALS AND METHODS SAMPLE COLLECTION AND PREPARATION Twenty (APC) or 25 (coliforms and E. coli) whole ready-to-cook broiler chicken carcasses were obtained from the chiller exit of a commercial broiler processing facility in each of three replicate trials for each bacterial test type. The carcasses were transported to the laboratory on ice and individually bagged in sterile polyethylene bags (3,000 cc O 2 at 22.8 C per m 2 per 24 h at 1 atm). Carcasses were randomly separated into five groups containing four (APC) or five (coliforms and E. coli) carcasses in each group. Four (APC) or five (coliforms and E. coli) carcasses out of the first group were sampled and analyzed immediately. The remaining four groups of carcasses were sampled and analyzed after temperature abuse (above refrigeration termperatures) at 37 C for 2, 4, 6, or 8 h. Carcasses were sampled by rinsing with 100 ml of sterile deionized water according to the procedure described by Cox et al. [13]. CONVENTIONAL MICROBIOLOGICAL METHODOLOGY Aerobic plate counts were conducted according to the method described by Swanson et al. [9] in the Compendium of Methods for the Microbiological Examination of Foods. After incubation for 48 h at 35 C, plates that contained between 25 and 250 colonies were counted. Total coliform and E. coli populations of bacteria were enumerated using the VRBA and the MPN methods described by Hitchens et al. [10], respectively, in the Compendium of Methods for the Microbiological Examination of Foods. For the MPN assays, instead of conducting IMViC tests to differentiate the bacterial species, bacteria were removed from the plate and confirmed using a Gram-negative card that was monitored using the Vitek Automicrobic System [14]. OPTICAL MEASUREMENTS To enumerate total viable counts using the BioSys optical system [1], 1 ml rinse fluid from each carcass sample was placed into the BioSys vial. The BioSys vial contained an agar plug in the bottom and 9 ml basal medium containing the following (per liter of deionized water): 5 g Bacto-peptone [16], 3 g yeast extract [16], and 1 g d-glucose [17]. The basal medium was autoclaved at 121 C (15 psi) for 15 min. A dye solution was prepared by dissolving 30 mg brom-cresol-purple [16] into 2 ml 100% ethanol. This mixture was placed into 8 ml sterile deionized water. The final mixture (10 ml) was filter-sterilized (2 µ) and added to 1 L basal medium. The brom-cresol-purple indicator in the total viable counts medium was allowed to migrate completely into the agar detection window. The vial was placed into the BioSys instrument and monitored at 35 C. The time required for bacterial metabolites to accumulate and exceed the detection threshold [approximately 10 5 to 10 6 cfu/ml medium] was recorded as a detection time (DT). To enumerate total coliform populations of bacteria using the BioSys optical system, 5 ml rinse fluid from each carcass sample was placed FIGURE 1. Regression line for aerobic plate count (APC) data plotted against the BioSys optical method detection times in hours for enumeration of total viable count from chicken carcasses. Log 10 cfu/ml = 7.48 0.44(DT); r = 0.9211; n = 60, where DT = detection time.

144 JAPR: Research Report FIGURE 2. Histogram analysis of total aerobic bacterial count data for chicken carcasses using the specification limit of 10,000 cfu/ml. Hatched data points below the cutoff value are within the cfu limit, and those exceeding the cutoff value are above the cfu limit. into the BioSys vial. The BioSys vial contained an agar plug in the bottom and 5 ml single strength coliform medium [14]. The medium was demonstrated to be appropriate for selection of coliform bacteria by Russell et al. [18]. The brom-cresol-purple indicator in the coliform medium with dextrose (CMD) was allowed to migrate completely into the agar detection window. The vial was placed into the BioSys instrument and monitored at 35 C. The DT were recorded in hours. To enumerate E. coli using the BioSys optical system, 5 ml rinse fluid from each carcass were placed into the BioSys vial. The BioSys vial contained an agar plug in the bottom and 5 ml double strength coliform medium [14] supplemented with 2% dextrose (CMD). This medium was reported to be excellent for selec- FIGURE 3. Histogram analysis of total aerobic bacterial count data for chicken carcasses using the specification limit of 100,000 cfu/ml. Hatched data points below the cutoff value are within the cfu limit, and those exceeding the cutoff value are above the cfu limit.

RUSSELL: RAPID ENUMERATION OF BACTERIA 145 FIGURE 4. Regression line for violet-red-bile agar (VRBA) coliform counts plotted against the BioSys optical method detection times for enumeration of coliforms from chicken carcasses. Log 10 cfu/ml = 6.74 0.56(DT); r = 0.9160; n = 75, where DT = detection time. tion of E. coli by Edmiston and Russell [19]. The brom-cresol-purple indicator in the CMD was allowed to migrate completely into the agar detection window. The vial was placed into the BioSys instrument and monitored at 42 C. The DT were recorded in hours. STATISTICAL ANALYSES The experimental design was a 4 (APC) or 5 (coliforms and E. coli) 3 5 of carcass, replication, and hour (temperature abuse time) for broiler chicken carcasses using a total of 60 chicken carcasses for APC and 75 chicken carcasses for coliforms and E. coli. All microbiological analyses were conducted in duplicate (except for BioSys) on each carcass rinse, averaged, and then transformed into log 10 values prior to statistical analyses. Linear regression was conducted using the General Linear Models procedure of SAS software [20]. Correlation coefficients (r) and line equations were generated for each test method by regressing the data against the standard APC, coliform, or MPN procedure. RESULTS AND DISCUSSION As bacteria multiply in microbiological growth media, they produce metabolites that accumulate. Eventually, these metabolites accumulate to the extent that they significantly change the chemical characteristics of the medium (ph or oxygen-reduction potential). Using an indicator dye in the growth medium that responds to these changes allows for detection of the change, because the color of the medium will change in accordance with the shift in ph FIGURE 5. Histogram analysis of coliform count data for chicken carcasses using the specification limit of 100 cfu/ml. Hatched data points below the cutoff value are within the cfu limit, and those exceeding the cutoff value are above the cfu limit.

146 JAPR: Research Report FIGURE 6. Histogram analysis of coliform count data for chicken carcasses using the specification limit of 1,000 cfu/ml. Hatched data points below the cutoff value are within the cfu limit, and those exceeding the cutoff value are above the cfu limit. or redox potential. The BioSys instrument has an incubation block that contains 32 sample ports. Each sample port contains a light emitting diode (LED) that is used to transmit light through the medium at the junction where the agar plug comes in contact with the medium. A detector is used to pick up the light on the other side of the detection window. The point at which this ph or redox shift occurs is known as the detection threshold and is a function of the medium, competing microorgan- isms, and the incubation temperature. Once this shift in the medium occurs, the time required for the shift to take place is recorded. This shift usually occurs when bacteria reach a level of 10 6 cfu/ml. The time that elapses between the initiation of recording until the impedance shift occurs is referred to as the DT. The regression line for data collected using the APC method plotted against data obtained using the BioSys optical method for enumeration of total aerobic populations of bacteria FIGURE 7. Histogram analysis of coliform count data for chicken carcasses using the specification limit of 10,000 cfu/ml. Hatched data points below the cutoff value are within the cfu limit, and those exceeding the cutoff value are above the cfu limit.

RUSSELL: RAPID ENUMERATION OF BACTERIA 147 FIGURE 8. Regression line for Escherichia coli most probable numbers plotted against the BioSys optical method detection times for enumeration of E. coli from chicken carcasses. Log 10 cfu/ml = 9.60 0.89(DT); r = 0.9111; n = 75, where DT = detection time. from chicken carcasses is presented in Figure 1. For chicken carcasses, the correlation coefficient for the regression line comparing APC with BioSys optical measurements was 0.9211. The line equation was as follows: log 10 cfu/ml = 7.48 0.44(DT). Using the Bio- Sys optical method, results were able to be obtained in 2 to 11 h, as opposed to the 48-h period required to enumerate APC using the standard method. Included with the BioSys instrument is software that can be used to set specification limits and to determine cut-off times. For example, if a company produced chicken for a restaurant chain, and the restaurant chain had a limit of 10,000 total aerobic bacteria per milliliter of chicken rinse, then this value may be inserted into the histogram program provided with the instrument to obtain a cut-off time. Specification limits of 10,000 and 100,000 have been inserted into this equation, and cut-off times have been generated and are presented in Figures 2 and 3, respectively. For the specification limit of 10,000 cfu/ml, the software generated a cut-off time of 8.0 h based on the calibration curve line equation (Figure 2). Using this information, the operator knows that any samples that result in detection prior to 8.0 h should be retained because they exceed the customers specification level of 10,000 cfu/ml rinse. For the specification limit of 100,000 cfu/ml, the software generated a cut-off time of 6.0 h based on the calibration curve line equation (Figure 3). The regression line for data from the VRBA coliform count method plotted against the Bio- Sys optical method for enumeration of total coliform populations of bacteria from chicken carcasses is presented in Figure 4. For chicken FIGURE 9. Histogram analysis of Escherichia coli count data for chicken carcasses using the specification limit of 100 cfu/ml. Hatched data points below the cutoff value are within the cfu limit, and those exceeding the cutoff value are above the cfu limit.

148 JAPR: Research Report FIGURE 10. Histogram analysis of Escherichia coli count data for chicken carcasses using the specification limit of 1,000 cfu/ml. Hatched data points below the cutoff value are within the cfu limit, and those exceeding the cutoff value are above the cfu limit. carcasses, the correlation coefficient for the regression line comparing coliform counts with BioSys optical measurements was 0.9160. The line equation was as follows: log 10 cfu/ml = 6.74 0.56(DT). As with APC, the BioSys could be used to enumerate coliforms in 3.5 to 10.5 h, as opposed to the 48-h period required to enumerate coliforms using the VRBA plating method. Specification limits of 100, 1,000, and 10,000 were inserted into the histogram program for coliforms, and cut-off times were generated and are presented in Figures 5, 6, and 7, respectively. For the specification limits of 100, 1,000, and 10,000 cfu/ml coliforms, the software generated cut-off times of 8.5, 6.5, and 6.0 h, respectively, based on the calibration curve line equations (Figures 5, 6, and 7, respectively). The regression line for data from the threetube MPN method plotted against the BioSys optical method for enumeration of generic E. coli from chicken carcasses is presented in Figure 8. The correlation coefficient for the regression line comparing E. coli MPN to BioSys optical measurements was 0.9111. Using the BioSys optical method, E. coli were able to be enumerated in 3.5 to 10 h, as opposed to the 8-d period required to enumerate E. coli using the MPN method. Using the BioSys optical method, more variance was associated with samples that contained lower numbers of E. coli. At higher numbers, the variance was not as noticeable. For enumerating E. coli, the histogram program may prove to be extremely useful. As mentioned previously, the U.S.D.A. specification limits for acceptable, questionable, and unacceptable levels of E. coli on poultry carcasses are <100, 100 to <1,000, and >1000 cfu/ml, respectively [8]. For the specification limits of 100 and 1,000 cfu/ml E. coli, the software generated cut-off times of 8.0 and 7.5 h, respectively, based on the calibration curve line equations (Figures 9 and 10, respectively). The Bio- Sys method would allow the industry to conduct E. coli counts and receive results on the day of processing in less than 8 h as a means of ensuring that products meet or exceed the USDA requirements as set forth in the Pathogen Reduction/HACCP Regulation. In summary, the BioSys optical method performed very well in comparison with traditional methods for enumerating APC, coliforms, and E. coli from chicken carcasses. The advantages to using the BioSys method are that results were able to be obtained in 2 to 11 h rather than the 48 h required to conduct APC or coliform counts or 8 d required to conduct the MPN procedure. These methods would allow processors to be able to test product and obtain results prior to shipping the product, avoiding the cost and loss of reputation associated with a recall

RUSSELL: RAPID ENUMERATION OF BACTERIA 149 or food-borne illness outbreak. Moreover, although this instrument has a capital cost associated with its purchase, the cost per test is much lower than the cost per test to conduct the standard methods. The labor and time required to prepare samples using the rapid method is much less as well, because only one dilution is required, as opposed to conducting multiple dilutions and pipetting to and reading multiple plates. This method should be beneficial to the poultry industry for rapidly enumerating APC, coliforms, and E. coli. CONCLUSIONS AND APPLICATIONS 1. The BioSys optical method would be less costly, less time-consuming, and more rapid for determining APC for broiler chicken carcasses. 2. The BioSys optical method would be a useful method for estimating coliform counts on broiler chicken carcasses. 3. Escherichia coli counts would be able to be conducted in hours using the BioSys method, allowing poultry companies to remain in compliance with the U.S.D.A. HACCP regulations. 4. The histogram program provided with the BioSys system allows the operator to determine easily whether samples are within specification limits. 1. BioSys, Inc., Ann Arbor, MI. 2. Archer, D.L., and J.E. Kvenberg, 1985. Incidence and cost of foodborne diarrheal disease in the United States. J. Food Prot. 48:887 894. 3. Bennett, J., S. Holmberg, M. Rogers, and S. Solomon, 1987. Infectious and parasitic diseases. Pages 102 114 in: Closing the Gap: The Burden of Unnecessary Illness. R. Amler and H. Dull, Eds. Oxford Univ. Press, New York, NY. 4. Council of Agricultural Science and Technology, 1994. Foodborne pathogens: risks and consequences. Task Force Report No. 122, Sept., Ames, IA. 5. Todd, E.C.D., 1989. Preliminary estimates of costs of foodborne disease in the United States. J. Food Prot. 52:595 601. 6. United States Department of Agriculture, Food Safety and Inspection Service, 1996. The Final Rule on Pathogen Reduction and Hazard Analysis and Critical Control Point (HACCP) Systems, Backgrounder, July, p. 1. 7. United States Department of Agriculture, Food Safety and Inspection Service, 1996. How USDA s new food safety system will fight bacteria that cause food borne illness. Key Facts Bulletin, July. 8. United States Department of Agriculture, Food Safety and Inspection Service, 1996. Pathogen reduction; Hazard Analysis and Critical Control Point (HACCP) systems; final rule. Fed. Reg. 61:38939 38944. 9. Swanson, K.M.J., F.F. Busta, E.H. Peterson, and M.G. Johnson, 1992. Colony count methods. Pages 75 95 in: Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington, D.C. 10. Hitchens A.D., P.A. Hartman, and E.C.D. Todd, 1992. Coliforms Escherichia coli and its toxins. Page 341 in: Compendium of Methods for the Microbiological Examination of Foods. REFERENCES AND NOTES C. Vanderzant and D. F. Splittstoesser, Eds. American Public Health Association, Washington, DC. 11. Shelef, L.A., and R. Firstenberg-Eden, 1997. Novel selective and non-selective optical detection of micro-organisms. Lett. Appl. Microbiol. 25:202 206. 12. Shelef, L.A., S. Mohammed, W. Tan, and M.L. Webber, 1997. Rapid optical measurements of microbial contamination in raw ground beef and effects of citrate and lactate. J. Food Prot. 60:673 676. 13. Cox, N.A., J.E. Thomson, and J.S. Bailey, 1981. Sampling of broiler carcasses for Salmonella with low volume water rinse. Poult. Sci. 60:768 770. 14. biomérieux, Inc., Hazelwood, MO. 15. Difco, Becton Dickinson, Sparks, MD, 21152. 16. Fisher Scientific, Norcross, GA, 30091. 17. Russell, S.M., D.L. Fletcher, and N.A. Cox, 1995. Comparison of media for determining temperature abuse of fresh broiler carcasses using impedance microbiology. J. Food Prot. 58(10):1124 1128. 18. Edmiston, A.L., and S.M. Russell, 1998. A rapid microbiological method for enumerating Escherichia coli from broiler chicken carcasses. J. Food Prot. 61(10):1375 1377. 19. SAS Institute, 1988. SAS /STAT Guide for Personal Computers. Version 6.03 Edition. SAS Institute Inc., Cary, NC. ACKNOWLEDGMENTS This study was supported in part by state and Hatch funds allocated to the Georgia Agricultural Experiment Station. Appreciation is extended to BioSys, Inc. for its generous support, which included the BioSys Microbiological System, technical assistance, and supplies.