PHILIPUS PANGLOLI, FELIX JACKSON, HAROLD A. RICHARDS, JOHN R. MOUNT and F. ANN DRAUGHON 1

COMPARISON OF CONVENTIONAL PLATING AND SIMPLATE METHODS FOR ENUMERATION OF AEROBIC MICROORGANISMS, COLIFORM AND ESCHERICHIA COLI IN FARM ENVIRONMENTAL SAMPLES PHILIPUS PANGLOLI, FELIX JACKSON, HAROLD A. RICHARDS, JOHN R. MOUNT and F. ANN DRAUGHON 1 Food Safety Center of Excellence Agricultural Experimental Station The University of Tennessee 2605 River Drive, Knoxville, TN 37996 Accepted for Publication February 24, 2006 ABSTRACT The objective of this study was to compare conventional plating to Sim- Plate methods for enumeration of aerobic microorganisms, coliforms and Escherichia coli in samples from farm animals and their environment. Samples evaluated included rectal swabs, fecal material, bedding, litter and feed and soil from beef and dairy cattle, swine and poultry farms. Standard method agar pour plating and conventional violet red bile agar with 4-methylumbelliferyl b-d-glucuronide were compared to the SimPlate total plate count-color indicator method for aerobic counts and the SimPlate coliform and E. coli color indicator method for total coliforms and E. coli counts, respectively. Overall, the SimPlate methods produced counts that were not significantly different (P 0.05) from those of the conventional methods. Population counts from the SimPlate methods were highly correlated with colony-forming unit counts from the conventional methods with r = 0.90, 0.94 and 0.94 for aerobic counts, total coliforms and E. coli, respectively. Thus, the data from this study indicate that SimPlate methods are comparable and reliable for enumeration of aerobic microorganisms, coliforms and E. coli in environmental farm samples. INTRODUCTION Aerobic microorganisms, coliforms and Escherichia coli are important indicators of sample quality and are frequently used in food science and dairy laboratories. Applications include monitoring fecal contamination in water, 1 Corresponding author. TEL: 865-974-7425; FAX: 865-974-2750; EMAIL: draughon@utk.edu Journal of Rapid Methods & Automation in Microbiology 14 (2006) 258 265. All Rights Reserved. 258 2006, The Author(s) Journal compilation 2006, Blackwell Publishing

RAPID METHODS FOR INDICATOR MICROORGANISMS 259 sanitary conditions in food processing plants and adequacy of heat application in food pasteurization processes (Morton 2001; Feng et al. 2002). High counts of fecal indicator bacteria may also be associated with the presence of pathogens (Kornacki and Johnson 2001); therefore, convenient methods for detection and enumeration are desirable. One such method, SimPlate, has distinct advantages over conventional plating methodologies. The SimPlate methods require less media preparation and eliminate uncertain counts caused by overcrowding, spreading colonies and interference of particulate matter on plating methods (Feldsine et al. 2003). Detection and enumeration of microorganisms by the SimPlate methods rely on a binary detection technology. Visible color changes occur as a result of bacterial enzyme interaction with substrates present in the culture medium (Townsend and Naqui 1998; Feldsine et al. 2003). These biochemical reactions allow for more sensitive enumeration because fewer microorganisms are required to cause the indicator color change than are needed to form a visible colony on agar plates. The SimPlate total plate count-color indicator (TPC-CI) is designed for enumeration of aerobic bacterial population in foods and has been approved by the Research Institute of the Association of Analytical Chemists (AOAC), Method 2002.7 (Feldsine et al. 2003). The SimPlate coliform and E. coli color indicator (CEc-CI) method is used for detection and quantification of coliforms and E. coli populations in foods. Several studies have indicated that the SimPlate CEc-CI method is comparable to other methods, and is a suitable alternative for detection and enumeration of total coliforms and E. coli in foods (Townsend et al. 1998; Russel 2000; Lee and Lewis 2003). The SimPlate CEc-CI method has recently been approved by the Research Institute of the AOAC, Method 2005.3 (Feldsine et al. 2005). Nevertheless, information on the efficacy of the SimPlate methods for use in environmental farm samples is limited. The objective of this study was to compare the SimPlate TPC-CI with the standard method agar (SMA) pour-plating methods for enumeration of aerobic microorganisms, and the SimPlate CEc-CI with the violet red bile agar containing 4-methylumbelliferyl b-d-glucuronide (VRB-MUG) plating methods for detection and quantification of coliforms and E. coli in various animal and environmental samples. The data collected for this study will suggest whether or not these methods are appropriate for use with environmental samples. MATERIALS AND METHODS Sample Collection and Preparation Samples evaluated included rectal swabs, animal feeds, soil, bedding and litter and feces from beef cattle, dairy, poultry and swine farms. Feed samples

260 P. PANGLOLI ET AL. included grass, total mixed ration (TMR), fresh feed (sampled prior to feeding) and trough feed (sampled from remains in the trough or feeder). Soils were sampled from grazing pasture on beef cattle farms and from around barns on dairy, poultry and swine farms. Bedding samples, a mixture of various materials, were collected from areas where animals usually rested on beef farms or from dairy barns. Duplicate samples were collected, transported overnight to the laboratory and stored at refrigerated temperature (4C) until analyzed. Swab samples were collected from four animals, stored and transported individually in Cary Blair agar culture swabs (Becton Dickinson, Sparks, MD). All samples were naturally contaminated. Test samples (25 g) and swabs were diluted in 0.1% peptone water to prepare 10-fold serial dilutions. Each of the experiments was conducted in triplicate. Enumeration of Aerobic Microorganisms Conventional SMA pour plating was used to enumerate aerobic microorganisms (Swanson et al. 2001). An aliquot (1.0 ml) of the dilution for each sample was plated, mixed with tempered SMA medium and incubated at 35C for 24 h. A 24-h incubation period was used as environmental farm samples often resulted in overcrowded plates after 48-h incubation because of spreaders. Results are reported as aerobic plate count (APC) or colony-forming unit per gram of sample or per swab. Enumeration of aerobic microorganisms with the SimPlate TPC-CI multiple test medium (BioControl System, Bellevue, WA) method was performed according to the manufacturer s instructions and procedures of Feldsine et al. (2003). Sterile water for rehydrating the SimPlate TPC-CI medium contained 1 ml of supplement A per 100 ml. After incubation at 35C for 24 h, the wells showing a color change from the background color were counted as positive wells. The total number of microorganisms per sample was determined by correlating the total number of positive wells with the corresponding microbial population in the SimPlate conversion table and by multiplying the count by the appropriate dilution factor. Enumeration of Coliforms and E. coli Coliforms and E. coli were identified and quantified using conventional pour plating on VRB-MUG (Difco, Sparks, MD) plates according to procedures of Kornacki and Johnson (2001). The plates were incubated at 35C for 24 h prior to counting coliforms (purple-red colonies) and E. coli (fluorescent colonies under long-wave UV light). Enumeration of coliforms and E. coli using the SimPlate CEc-CI method with multiple test medium (BioControl System) was performed according to the manufacturer s instructions and procedures of Feldsine et al. (2005). The total number of positive wells for

RAPID METHODS FOR INDICATOR MICROORGANISMS 261 coliforms was counted on the basis of color change. Wells were counted positive for E. coli on the basis of color change and fluorescence under UV light. The coliform and E. coli populations were determined on the basis of the number of positive wells correlated with the SimPlate conversion table, which generated a most probable number (MPN) per gram of sample or per swab. Statistical Analysis Data were subjected to analysis of variance with a completely randomized block design with nested treatment arrangement, blocked on replication. Samples were nested in the farm as the samples collected from each farm category were different. The data were analyzed with SAS mixed procedures using SAS software release 9.0 (SAS Institute Inc., Cary, NC). Significant differences among means were determined by the least square means method with the P value differences option. Correlation coefficients were generated using Microsoft Office Excel version 2000 (Microsoft Corporation, Redmond, WA). RESULTS AND DISCUSSION Generally, the total APC was higher for a given sample in the SimPlate TPC-CI method compared with the conventional SMA method (Table 1). However, this difference was only significant (P 0.05) in two specific types of sample. The APCs of trough TMR and bedding samples from dairy farms were 1.5 2.0 orders of magnitude higher or lower using the SimPlate method. Data obtained in this study with farm and environmental samples are comparable to previous research comparing SimPlate TPC-CI to SMA plating counting in foods (Beuchat et al. 1998; Feldsine et al. 2003). The SimPlate CEc-CI method tended to produce higher coliform and E. coli counts for the collected samples than estimates derived from conventional VRB-MUG plating (Table 2). In general, these differences were not significant (P 0.05); however, the SimPlate method produced significantly higher (P 0.05) estimates of either coliform or E. coli counts for five specific sample types. Rectal swabs from beef cattle resulted in higher counts of both coliforms and E. coli when comparing the methodologies, perhaps indicating that Sim- Plate CEc-CI is more sensitive for this sample type. Bedding materials from dairy farms had significantly higher coliform counts and soil from beef cattle farms had significantly higher E. coli counts when using SimPlate estimates. This may be due to the variety of dairy bedding materials used across the U.S.A., and caution is recommended if SimPlate is being used for dairy bedding or TMR samples. An unexpected result was obtained from analysis of grass clippings

262 P. PANGLOLI ET AL. TABLE 1. COMPARISON OF THE STANDARD METHOD AGAR (SMA) AND THE SimPlate TOTAL PLATE COUNT-COLOR INDICATOR METHOD FOR ENUMERATION OF AEROBIC MICROORGANISMS IN ANIMAL AND ENVIRONMENTAL FARM SAMPLES (LOG cfu/g OR SWAB)* Farm/sample Aerobic plate count SE SMA SimPlate Beef cattle Swabs 8.0 8.8 0.7 Grass 7.8 7.7 0.6 Pasture soil 6.6 6.9 0.6 Bedding 6.3 7.3 0.6 Dairy Swabs 8.6 9.1 0.7 Fresh TMR 4.0 5.4 0.7 Trough TMR 5.3a 6.8b 0.7 Soil 5.9 5.8 0.6 Bedding 8.0a 5.9b 0.6 Poultry Swabs 8.3 8.4 1.0 Fresh feed 6.0 5.6 0.9 Trough feed 6.7 6.7 0.9 Soil 6.0 6.9 0.8 Litter 9.0 8.7 0.8 Swine Swabs 8.2 8.5 0.7 Fresh feed 4.6 4.5 0.7 Trough feed 5.6 5.3 0.7 Soil 4.6 4.8 0.6 Feces 7.2 6.6 0.6 *Means followed by different letters are significantly different (P 0.05). TMR, total mixed ration containing mainly silage and grains. collected from grazing pastures. For this sample type, and only in this sample, the conventional VRB-MUG method produced higher counts of E. coli than SimPlate, suggesting that grass clippings may complicate or inhibit the Sim- Plate methodology. Data obtained from this study are similar to previous research, which indicated that the SimPlate method consistently produced higher and more sensitive coliform and E. coli counts in food samples than VRB-MUG plating (Townsend et al. 1998; Lee and Lewis 2003). Using conventional plating methodology to enumerate aerobic microflora in environmental samples is problematic, because these samples tend to contain high levels of bacteria, fungi and large quantities of particulate matter.

RAPID METHODS FOR INDICATOR MICROORGANISMS 263 TABLE 2. COMPARISON OF CONVENTIONAL VIOLET RED BILE AGAR WITH 4-METHYLUMBELLIFERYL b-d-glucuronide (VRB-MUG) METHOD WITH THE SimPlate COLIFORM AND ESCHERICHIA COLI COLOR INDICATOR FOR ENUMERATION OF COLIFORMS AND E. COLI OF ANIMAL AND ENVIRONMENTAL FARM SAMPLES (LOG POPULATIONS/g OR SWAB)* Farm/sample Coliform E. coli VRB-MUG SimPlate SE VRB-MUG SimPlate SE Beef cattle Swabs 8.0a 8.8b 0.6 7.8a 8.8b 0.6 Grass 6.0 6.6 0.6 4.5a 3.1b 0.6 Pasture soil 5.0 5.1 0.6 2.8a 3.7b 0.6 Bedding 3.9 4.1 0.6 2.8 3.2 0.6 Dairy Swabs 8.6 9.1 0.6 7.7a 8.7b 0.6 Fresh TMR 2.8 2.5 0.7 2.8 3.1 0.7 Trough TMR 3.4 3.2 0.7 2.9 3.6 0.7 Soil 4.0 3.8 0.6 3.8 3.2 0.6 Bedding 4.5a 6.8b 0.6 4.0 4.5 0.6 Poultry Swabs 7.4 8.1 0.9 7.4 8.1 0.9 Fresh feed 4.2 3.5 0.8 2.5 2.5 0.8 Trough feed 4.1 4.4 0.8 2.5 2.5 0.8 Soil 4.1 4.0 0.7 3.6 3.7 0.7 Litter 4.7 5.1 0.7 3.8 4.4 0.7 Swine Swabs 8.6 8.9 0.6 8.3 8.9 0.6 Fresh feed 3.2 3.7 0.7 2.4 2.1 0.7 Trough feed 2.5 3.1 0.7 2.0 2.1 0.7 Soil 3.6 3.1 0.6 1.9 2.2 0.6 Feces 4.3 4.7 0.6 4.0 4.6 0.6 *Means within the same group of microorganism followed by different letters are significantly different (P 0.05). TMR, total mixed ration containing mainly silage and grains. These factors contribute to chronic undercounting because of difficulties in distinguishing single colonies on a plate. The SimPlate methodology offers an enticing alternative that may result in more accurate estimates. In this study, SimPlate produced consistently higher counts of aerobic microorganisms, coliforms and E. coli. This result is probably due to the fact that the SimPlate conversion table utilized the same mathematical principles as the 3- or 5-tube MPN method (Townsend and Naqui 1998). However, the SimPlate estimates are likely to be more accurate and more highly correlated to plate-counting estimates because the design of the plate incorporates a much larger sample set, 84 or 198 test wells per plate. In fact, the data presented here indicate that

264 P. PANGLOLI ET AL. the SimPlate methods are highly correlated to the conventional SMA and VRB-MUG methodologies, which is evidenced by relatively high coefficient correlations for estimates of aerobic microorganisms (0.90), coliforms (0.94) and E. coli (0.94). Therefore, the SimPlate methods are accurate and reliable tools for estimating coliforms and aerobic microflora from environmental samples with the exceptions noted. Note that SimPlate is an MPN-based method with all the advantages and disadvantages common to MPN methodologies. However, the ease of use and reliability of SimPlate should make it a desirable tool for environmental microbiology research. ACKNOWLEDGMENTS Funding for this study was provided by the National Alliance for Food Safety and Security, the University of Tennessee Food Safety Center of Excellence and the U.S. Department of Agriculture National Research Initiative. REFERENCES BEUCHAT, L.R., COPELAND, F., CURIALE, M.S., DANISAVICH, T., GANGAR, V., KING, B.W., LAWLIS, T.L., LIKIN, R.O., OKWUSOA, J., SMITH, C.F., ET AL. 1998. Comparison of the SimPlate total plate count method with Petrifilm, and conventional pour-plate methods for enumerating aerobic microorganisms in foods. J. Food Prot. 61, 14 18. FELDSINE, P.T., LEUNG, S.C., LIENAU, A.H., MUI, L.A. and TOWNSEND, D.E. 2003. Enumeration of total aerobic microorganisms in foods by SimPlate total plate count-color indicator methods and conventional culture methods: Collaborative study. J. AOAC Int. 86, 259 274. FELDSINE, P.T., LIENAU, A.H., ROA, N.H. and GREEN, S.T. 2005. Enumeration of total coliforms and E. coli in foods by the SimPlate coliform and E. coli color indicator method and conventional culture methods: Collaborative study. J. AOAC Int. 88, 1318 1333. FENG, P., WEAGANT, S.D. and GRANT, M.A. 2002. Enumeration of Escherichia coli and coliform bacteria. In U.S. Food and Drug Administration. Bacteriological analytical manual online. http://www. cfsan.fda.gov/~ebam/bam-4.html (accessed July 13, 2006). KORNACKI, J.L. and JOHNSON, J.L. 2001. Enterobacteriaceae, coliforms, and Escherichia coli as quality and safety indicators. In Compendium Methods for the Microbiological Examination of Foods, 4th Ed. (F.P. Downes and K. Ito, eds.) pp. 69 82, American Public Health Association, Washington, DC.

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