3 Petrifilm Environmental Listeria Plate for the Rapid Enumeration of Listeria from Environmental Surfaces

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1 3 Petrifilm Environmental Listeria Plate for the Rapid Enumeration of Listeria from Environmental Surfaces Barbara Horter and Henry Lubrant, 3M Microbiology, St. Paul, MN, USA Abstract Accurate and rapid detection of Listeria is of significant interest to the food industry. The 3M Petrifilm Environmental Listeria Plate has been developed in order to provide a Listeria result from environmental samples within 27 to 31 hours. This study compared the Petrifilm Plate method with ISO using PALCAM agar for detection and enumeration of Listeria. Studies of 88 strains demonstrated that the Petrifilm Environmental Listeria Plate has an inclusivity (sensitivity) of 98% and an exclusivity (specificity) of 100% when compared to ISO When comparing colony counts of the strains tested, a paired t-test showed that there was no significant difference between counts on the Petrifilm Plate method and the ISO method (p>0.05). Three different surfaces and nine different collection devices were tested using both methods. The surfaces were artificially inoculated with Listeria and background microbiota. Both sponges and swabs were used as collection devices. After a 60 to 75 minute repair step in buffered peptone water at room temperature (23.7 C), the samples were plated onto Petrifilm Plates and onto PALCAM agar plates. Results showed that, on average, the Petrifilm Plate method was not statistically different (p>0.05) from the ISO method for enumeration of Listeria. Introduction Keeping Listeria out of the food-manufacturing environment is the best defense against product contamination. A vigorous environmental monitoring program is a vital preventative measure in the management of product contamination due to Listeria. The 3M TM Petrifilm TM Environmental Listeria Plate was developed to provide rapid and quantitative Listeria results from environmental samples. The Petrifilm Environmental Listeria Plate is a sample-ready culture medium system containing selective agents, nutrients, a cold-water-soluble gelling agent, and a chromogenic indicator. Petrifilm Environmental Listeria Plates are used for the detection and/or enumeration of Listeria in environmental samples. The sample is collected using a sponge, swab or other pre-moistened sample device. After five milliliters of repair broth - buffered peptone water - is added to the sample, the sample is mixed and then allowed to sit at room temperature for a minimum of 1 hour and up to a maximum of 1.5 hours. The sample is mixed and 3 milliliters is plated onto the Petrifilm Plate. The 1 3Microbiology

2 gelling agent is allowed to solidify after inoculation, and the plate is then incubated for hours at C or C. Red-violet colonies on the plate are Listeria (see Figure 1). Figure 1. Petrifilm Environmental Listeria Plate showing red-violet Listeria colonies There were 2 objectives of this study. One was to compare the Petrifilm Environmental Listeria Plate method with the ISO method for inclusivity of target organisms and exclusivity of non-target organisms. The second objective was to compare the Petrifilm Environmental Listeria Plate method with the ISO method for the detection and enumeration of Listeria from different surface materials. Materials and Method Strain Testing Bacteria for the study 51 Listeria spp. and 37 non-listeria including Bacillus, Brevibacterium, Enterococcus, Erysipelothrix, Escherichia, Kurthia, Lactobacillus, Pediococcus, Pseudomonas, Staphylococcus and Streptococcus were selected from the 3M culture collection or were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Frozen cultures were stored at -84 o C in 15% glycerol. Strains were tested according to the methods outlined in Figure 2. 2

3 Figure 2. Strain testing procedure Grow organisms overnight in TSB about 19 h Dilute organism in Standard Methods Buffer Petrifilm Plate Method ISO Method Add 0.5mL organism to 5 ± 0.5mL BPW Add 0.5ml organism to 4.5 ± 0.5mL BPW 1 hr ± 5 min at 25 ± 2 C 1 hr ± 5 min at 20 ± 2 C 3mL on Petrifilm Listeria Plate 1mL on PALCAM 150mm plate 1mL on PALCAM 150mm plate 28 ± 2 h at 35 ± 1 C h at 37 ± 1 C Red-violet colonies are Listeria Perform confirmation tests Calculate Colony Counts 3 Record colony counts

4 Surface Testing To simulate environmental testing, 45 samples were analyzed which consisted of 3 different surface types, 9 different collection devices and 5 different organisms. Samples were tested using the Petrifilm Environmental Listeria Plate and the ISO method. Surfaces stainless steel glazed earthen tile plastic (polypropylene) Collection devices Hydrasponge TM with 10 ml of neutralizing buffer (International Bioproducts) Hydrasponge TM with 10 ml of letheen broth (International Bioproducts) Hydrasponge TM with 10 ml of buffered peptone water (International Bioproducts) Spongesicle TM with 10 ml neutralizing buffer (International Bioproducts) dry, sterile sponge to which 10 ml of sterile water was added Quick Swab (3M) cotton-tipped swab with wooden shaft (Puritan) dacron-polyester-tipped swab with plastic shaft (Puritan), foam-tipped swab with plastic shaft (VWR) Organisms used in surface testing 2 Listeria monocytogenes Listeria innocua Listeria welshimeri E.coli Procedure Organisms were grown overnight in trypticase soy broth + 0.6% yeast extract. A 4 X 4 inch square area on each sterilized surface was inoculated with organisms a Listeria plus E.coli as background or E.coli by itself. The surfaces were allowed to dry overnight. Pre-moistened sampling devices were used to collect samples by swabbing or sponging the area in both a vertical and horizontal direction. The sampling device was returned to the container it came in or to a sterile test tube and 8 ml of buffered peptone water was added. The sample was either vortexed or stomached. The sample was allowed to sit at room temperature (23.7 C) for 60 to 75 minutes. Just before plating, the samples were remixed. A 3 ml sample was plated onto the Petrifilm Environmental Listeria Plate. One-third of a ml was plated onto each of 9 PALCAM agar plates. 4

5 Petrifilm Plates were incubated for 28 ± 0.5 h and PALCAM plates were incubated for 48 h at 35 ± 1 C. Colony counts were recorded. Data Analysis Inclusivity and exclusivity values were calculated for the Petrifilm Environmental Listeria Plate method as compared to the ISO method. For both the strain testing and the surface testing, raw counts were converted to log 10 counts to more nearly match the underlying assumption of normality. A paired t-test was used to compare the Petrifilm Environmental Listeria Plate method with the ISO method for each portion of the study. Results Strain Testing The inclusivity and exclusivity of the Petrifilm Environmental Listeria Plate method as compared to the ISO method is listed below. Testing was performed on 51 Listeria isolates and 37 non-listeria isolates. The results are shown in Table 1. The following organisms did not grow on either method: Listeria seeligeri ATCC 35967, Listeria grayi ATCC , Listeria monocytogenes ATCC and ATCC J Table 1. Identification of Listeria organisms by the Petrifilm Plate and ISO methods. ISO Petrifilm Environmental Listeria Plate Inclusivity (Sensitivity) = 98% Exclusivity (Specificity) = 100% Table 2 shows the results of the comparison between the Petrifilm Environmental Listeria Plate method and the ISO method for colony counts from strain testing. 5

6 SE c t-value d p-value d Table 2. Comparison of the Petrifilm Plate method with the ISO method for the enumeration of Listeria. Testing N a Mean log Difference b Strains Surfaces a Number of samples used in the analysis b mean log difference: log ISO count-log Petrifilm Plate count c Standard error of the mean log difference d Value associated with the statistical test The mean log Listeria counts from the Petrifilm Plate method were not significantly different from the mean log counts from the ISO method. Surface Testing Results from the comparison between the Petrifilm Environmental Listeria Plate method and the ISO method for colony counts on surface testing are listed in Table 2. The mean log Listeria counts were not significantly different when comparing the log colony counts from the Petrifilm Environmental Listeria Plate method to the log colony counts of the ISO method for surface testing Conclusion Results with the Petrifilm Environmental Listeria Plate method were similar to those of the ISO method for strain and surface testing. The Petrifilm Plate gave results in less than one-third of the time of the reference method. This new Petrifilm Plate method provides the added advantages of a convenient, space-saving and waste-reducing, sample-ready medium. REFERENCES ISO :1998 (E), Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria monocytogenes Part 2: Enumeration method (1998) International Organization for Standardization, Geneva, Switzerland. AKNOWLEDGEMENTS The authors wish to thank Kathryn Lindberg for statistical consulting. Hydrasponge TM and Spongesicle TM are trademarks of International Bioproducts 3M Microbiology 3M Center, Bldg E-05 St. Paul, MN USA microbiology@mmm.com 6 Petrifilm is a trademark of 3M. 3M 2004

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