MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2,

MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 355 FOOD BIOLOGICAL CONTAMINANTS Sensitivity and Specificity of the Sanita-kun Aerobic Count: Internal Validation and Independent Laboratory Study HIROSHI MORITA Chisso Corp., 13-1 Kachidoki 3 Chome, Chuo-ku, Tokyo 104-8555, Japan MASASHI USHIYAMA, SHIGEYUKI AOYAMA, and MIHOKO IWASAKI Chisso Corp. Yokohama Research Center, 5-1 Okawa Kanazawa-ku, Yokohama 236-8605, Japan The Sanita-kun Aerobic Count consists of a transparent cover film, an adhesive sheet, a layer of nonwoven fabric, and a water-soluble compound film, including a culture medium formula for detection of aerobic microorganisms. The Sanita-kun sheet was validated for 14 food categories in an internal study and an independent study was conducted on ground beef and hot dogs. Both studies showed no significant difference in performance between 5 or 8 replicates of the Sanita-kun sheets and AOAC Method 966.23, excluding some lots of foods. The correlation coefficient to plate count in the internal accuracy study was 0.99. The average relative standard deviation for repeatability of total foods was 0.26 and 0.19, respectively, excluding <10 average counts. The ruggedness study, which examined the influence of incubation temperature and period, recommended incubation of the Sanita-kun sheet at 32.5 2.5 C for 46 2h. Comparison of 3 lots of Sanita-kun sheets showed no decrease of performance in the older lot. The shelf-life of the sheet is at least 14 months. The Sanita-kun Aerobic Counts has been granted AOAC Performance Tested Method SM status. Performance Tested Method SM certification is a validation program administered by the AOAC Research Institute (AOAC RI). The goal of certification is to validate the performance claims of commercial test kits. Data supporting test kit performance claims, product literature, labels, manufacturing specifications, and QA/QC procedures are submitted by the kit manufacturer. A laboratory study is designed by AOAC RI and conducted at an independent facility. This report details the internal evaluation conducted by the manufacturer and the independent laboratory study of the Sanita-kun Aerobic Count (Chisso Corp., Tokyo, Japan) performed at Silliker Laboratory Corporate Research Center (South Holland, IL). Received March 4, 2002. Accepted by AH August 14, 2002. Corresponding author s e-mail: h.morita@chisso.co.jp. The total aerobic count indicates the level of microorganisms in food. The official standard method for determining total aerobic count uses plate count, which is prepared as follows: dissolve the powder of plate count medium, sterilize, cool, store at 45 1 C, add to Petri dish, mix, and solidify. It is time-consuming and labor-intensive, and results in bulky waste. Sanita-kun (Figure 1), a ready-to-use sheet medium which features an easy test for food and environment is an easy swabbing test. It is quantitative, space saving, produces little waste, and was developed as an alternative to plate count. The Sanita-kun Aerobic Count device is a cultural medium for the enumeration of aerobic bacteria. It consists of a nonwoven fabric on which a layer of microbial nutrients is deposited in a film. The nutrient film, consisting of a water-soluble polymer, contains nutrients for microorganisms and 2,3,5-triphenyltetrazolium chloride (TTC), which is reduced by bacterial metabolism to produce a visible red dye. The nonwoven fabric and nutrient film are constructed on a base film (Figure 2). The basic device (fabric, nutrient, and base film) is inserted between an adhesive backing to stabilize the basic device and a transparent film. The adhesive backing and the transparent top film serve to deter evaporation and facilitate inspection of the inoculated device. Sample solutions containing bacteria are deposited on the fabric portion of the device. The sample solution then diffuses throughout the entire pad to dissolve and release the nutrient compound film, forming a highly viscous solution. Bacteria will not permeate into the nutrient film layer because of its high viscosity. During the assimilation process of the dissolved nutrient film and the nonwoven fabric, bacteria migrate to or near the surface of the fabric, where they metabolize the nutrients and TTC to produce a visible red dye after incubation (Figure 2). METHOD Media and Reagents (a) Sanita-kun Aerobic Count plate. Chisso Corp. (Tokyo, Japan). (b) Butterfield s phosphate buffer (BPB). Dissolve 34 g KH 2 PO 4 in 500 ml water, adjust ph to 7.2 with 1N NaOH KH 2 PO 4 34 g, and bring volume to 1000 ml with distilled water. Autoclave 15 min at 121 C. Dilute 1.25 ml of above solu-

356 MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 Figure 1. sheet. tion and bring volume to 1000 ml with distilled water. Autoclave 15 min at 121 C. (c) Physiological saline. Dissolved 9.0 g NaCl in 1000 ml water and autoclaved 15 min at 121 C. Apparatus Sanita-kun Aerobic Counts package and (a) Incubator. For maintaining 32.5 2.5 C. (b) Blender. Waring, or equivalent, multispeed model and 500 ml or 1 L metal blender jars with covers. (c) Stomacher. IUL Instruments (Barcelona, Spain) Masticater Type S, or equivalent with stomacher bags. (d) Pipets. Gilson (Villier-le-Bel, France) Pipetman, or equivalent, with sterilized or autoclaved chips. Reading and Interpreting Results One red colony on the Sanita-kun sheet is counted as an aerobic colony. Colonies with a dim red color are also counted as aerobic colonies. When the number of red colonies is <300, one red colony is counted as 1 CFU. The CFU/g sample is determined by multiplying colonies by the dilution number. When the number of colonies is between 300 and 1000, colonies can be counted in the entire area or just one part. Internal Laboratory Study Precision (a) Repeatability. Several lots of 14 food types (frozen or raw pork, cabbage, roast pork, frozen or raw shrimp, ice cream, fried egg, red pepper, buckwheat flour, raisins, udon noodles, peanut butter, milk chocolate chips, dry cat food, and frozen pizza) were obtained at local retail markets. The contamination level of each sample was determined by AOAC Official Method 966.23. Several lots of each food type were mixed to prepare 4 target levels (<10, 10 25, ca 100, and ca 250 CFU/mL). Each subsample (50 g) was homogenized with 450 ml sterile physiological saline and diluted with sterile physiological saline. Portions (1 ml) of homogenates were inoculated to duplicate Sanita-kun and incubated at 35 C for 48 h. Red colonies formed on the sheets were counted, and standard deviations were calculated among 5 subsamples. (b) Accuracy. At least 3 lots each of 16 foods in 14 categories (frozen or) raw pork, cabbage, roast pork, frozen and raw shrimp, ice cream, shelled eggs, red pepper, oregano, buckwheat flour, raisins, udon noodles, peanut butter, milk chocolate chips, dry cat food, frozen pizza, and frozen gyoza Sample Preparation Portions of 50 or 25 g sample are added to a stomacher bag or blender cup; 450 or 225 ml BPB or sterile physiological saline is then added to the stomacher bag or blender cup and stomached or homogenized for 2 min. Sample homogenate is diluted with sterile physiological saline or BPB with decimal dilutions. Analysis Transparent cover film is opened, and 1 ml sample homogenate or dilutions (1:100, 1:1000, 1:10 000 is applied to the nonwoven fabric with pipet or equivalent. Transparent film is replaced to cover sample and will adhere to the sheet. The sheet is patted to ensure attachment; however, it should not be squeezed. The sheet is incubated for 46 2 h at 32.5 2.5 C. Red colonies are counted with a marking pen or colony counter. Used sheets are sterilized by autoclaving or boiling in hot water. Figure 2. Structure of basic device of Sanita-kun sheet.

MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 357 Table 1. Precision study Table 1. (continued) Target level, CFU/mL Average count, a CFU SD b RSD r Target level, CFU/mL Average count, a CFU SD b RSD r Pork <10 6 2 0.3 10 25 17 3 0.2 ca 100 118 15 0.13 ca 250 213 11 0.05 Cabbage <10 3 1 0.3 10 25 16 4 0.3 ca 100 107 23 0.21 ca 250 372 45 0.12 Frankfurter <10 5 1 0.2 10 25 23 5 0.2 ca 100 105 13 0.12 ca 250 288 16 0.06 Shrimp <10 2 1 0.5 10 25 9 3 0.3 ca 100 50 11 0.22 ca 250 141 25 0.18 Ice cream <10 4 1 0.3 10 25 25 1 0.04 ca 100 No sample ca 250 No sample Fried egg (Atsuyaki-tamago) <10 1 1 1.0 10 25 2 1 0.5 ca 100 24 5 0.2 ca 250 187 10 0.05 Red pepper <10 5 3 0.6 10 25 23 6 0.3 ca 100 108 20 0.19 ca 250 264 36 0.14 Buckwheat flour <10 5 2 0.4 10 25 29 14 0.48 ca 100 91 26 0.29 ca 250 230 68 0.30 Raisins <10 3 1 0.3 10 25 6 2 0.3 ca 100 No sample ca 250 No sample Udon noodles <10 3 1 0.3 10 25 23 10 0.43 ca 100 118 14 0.12 ca 250 360 34 0.09 Peanut butter <10 8 2 0.3 10 25 No sample ca 100 No sample ca 250 No sample Milk chocolate chips <10 8 2 0.3 10 25 34 12 0.35 ca 100 No sample ca 250 No sample Dry cat food <10 6 2 0.3 10 25 24 4 0.2 ca 100 124 16 0.13 ca 250 289 23 0.08 Frozen pizza <10 5 1 0.2 10 25 16 4 0.3 ca 100 69 17 0.24 ca 250 173 21 0.12 All foods <10 5 0.38 10 25 19 0.30 ca 100 91 0.19 ca 250 229 0.12 a b Average of counts of 5 subsamples. SD = Standard deviation of counts of 5 subsamples. meat dumpling were obtained at local retail markets. Each 50 g sample was homogenized with 450 ml BPB according to AOAC Official Method 966.23 except for shelled eggs, which were prepared according to AOAC Official Method 940.37. Sample dilutions (1:100, 1:1000, 1:10 000) were prepared with sterile physiological saline. Samples of 1 ml homogenate and dilutions were plated on 5 replicates of Sanita-kun sheets according to package instruction, and with plate count according to Method 966.23. After incubation at 35 C for 48 h, colonies formed on the plates, and Sanita-kun sheets were counted. (c) Ruggedness. In a comparison of 24 and 48 h incubation times, each 50 g sample was homogenized with 450 ml BPB according to AOAC Official Method 966.23 except shelled eggs, which were prepared according to AOAC Official Method 940.37. Sample dilutions (1:100, 1:1000, 1:10 000) were prepared by adding sterile physiological saline. Portions (1 ml) of sample homogenate and dilutions were applied on 5 replicate Sanita-kun sheets and incubated at 35 C for 24 h. Colonies were counted after incubation. The Sanita-kun sheets were then incubated again at 35 C for 24 h, totaling 48 h. Colonies were counted again. In an experiment to determine the effect of incubation temperature and time, each 50 g sample was added to a stomacher bag. To each bag, 450 ml sterile physiological saline was added, and the bag was stomached for 2 min. Sample dilutions (1:100, 1:1000, 1:10 000) were prepared by adding sterile physiological saline. Portions (1 ml) of sample homogenate and di-

358 MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 Table 2. Accuracy study Average count, a CFU RSD r APC, log 10 CFU/g Dilution Sanita-kun Sanita-kun Sanita-kun Frozen pork 1:1000 256 a 237 0.04 0.03 5.41 5.38 1:10000 27 22 0.14 0.07 Frozen pork 1:10000 119 114 0.08 0.12 6.08 6.04 1:100000 10 12 0.36 0.46 Frozen pork 1:10000 68 a 53 0.12 0.27 5.88 5.72 1:100000 7 5 0.31 0.60 Frozen pork 1:10000 189 b 275 0.15 0.09 6.28 6.43 1:100000 16 a 26 0.25 0.24 Raw pork 1:100000 109 a 123 0.07 0.05 7.04 7.08 1:1000000 10 13 0.43 0.19 Raw pork 1:10000 364 417 0.04 0.12 6.57 6.62 1:100000 45 43 0.09 0.12 Cabbages 1:10000 47 b 116 0.18 0.13 5.65 6.04 1:100000 3 9 0.80 0.40 Cabbages 1:10000 150 168 0.11 b 0.02 6.18 6.23 1:100000 14 16 0.46 0.28 Cabbages 1:10000 136 222 0.11 0.06 6.15 6.34 1:100000 14 24 0.22 0.24 Roast pork 1:1000 94 157 0.18 0.07 4.97 5.20 1:10000 9 15 0.27 0.31 Roast pork 1:100 98 143 0.16 0.07 4.04 4.15 1:1000 25 16 0.37 0.10 Roast pork 1:1000 19 50 0.21 0.11 4.28 4.69 1:10000 1 4 0.37 0.61 Frozen shrimp 1:100 107 104 0.10 0.08 4.04 4.00 1:1000 10 11 0.40 0.13 Frozen shrimp 1:100 30 38 0.20 0.16 3.46 3.57 1:1000 2 3 0.80 0.30 Frozen shrimp 1:100 124 a 140 0.07 0.06 4.08 4.15 1:1000 11 11 0.21 0.15 Frozen shrimp 1:1000 42 b 75 0.04 b 0.18 4.61 4.86 1:10000 2 b 6 0.56 0.27 Frozen shrimp 1:1000 29 b 84 0.19 0.17 4.46 4.91 1:10000 2 b 7 0.59 0.14 Frozen shrimp 1:1000 50 b 79 0.15 0.10 4.67 4.89 1:10000 2 a 7 0.71 0.41 Ice cream 1:10 57 83 0.08 0.19 2.79 2.94 1:100 11 14 0.34 0.36 Ice cream 1:10 32 27 0.17 0.10 2.52 2.45 1:100 5 3 0.27 0.41 Ice cream 1:10 33 35 0.21 0.20 2.52 2.53 1:100 3 2 0.26 0.70 Shelled eggs 1:10 157 176 0.13 0.06 3.18 3.23 1:100 13 15 0.24 0.23

MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 359 Table 2. (continued) Average count, a CFU RSD r APC, log 10 CFU/g Dilution Sanita-kun Sanita-kun Sanita-kun Shelled eggs 1:10 33 33 0.19 0.19 2.48 2.48 1:100 3 3 0.62 0.34 Shelled eggs 1:10 91 b 127 0.13 0.11 2.96 3.11 1:100 10 13 0.45 0.30 Oregano c 1:1000 116 112 0.04 0.13 5.04 5.04 1:10000 6 9 0.46 0.36 Oregano c 1:10000 113 135 0.07 0.13 6.04 6.11 1:100000 10 11 0.42 0.39 Oregano c 1:10000 46 a 61 0.09 0.14 5.66 5.79 1:100000 7 8 0.25 0.28 Red pepper 1:1000 88 77 0.07 0.19 4.94 4.89 1:10000 8 8 0.40 0.27 Red pepper 1:1000 90 87 0.15 0.14 4.96 4.94 1:10000 12 10 0.29 0.13 Red pepper 1:1000 94 89 0.08 0.08 4.99 4.95 1:10000 14 10 0.15 0.32 Buckwheat flour 1:1000 89 80 0.09 0.32 4.95 4.92 1:10000 9 11 0.32 0.31 Buckwheat flour 1:1000 74 77 0.10 0.19 4.87 4.88 1:10000 8 7 0.11 0.17 Buckwheat flour 1:1000 75 68 0.10 0.08 4.88 4.83 1:10000 8 7 0.28 0.23 Dry green laver 1:10 193 204 0.05 0.11 3.32 3.34 1:100 41 36 0.15 0.16 Raisins 1:100 31 22 0.31 b 0.10 3.49 3.38 1:1000 2 4 0.54 0.34 Raisins 1:100 15 a 18 0.07 0.09 3.18 3.28 1:1000 2 2 0.71 0.50 Raisins 1:100 5 8 0.45 0.46 2.64 2.88 1:1000 0 1 2.24 1.05 Udon noodles 1:1000 11 14 0.23 0.29 4.04 4.11 1:10000 1 0 0.56 2.24 Udon noodles 1:1000 236 b 331 0.17 a 0.04 5.38 5.52 1:10000 26 29 0.12 0.10 Udon noodles 1:100 42 46 0.15 0.12 3.62 3.66 1:100 4 4 0.39 0.50 Peanut butter 1:10 3 3 0.39 0.53 <2.34 <2.34 1:100 0 0 2.24 2.24 Peanut butter 1:10 7 a 12 0.58 0.13 <2.34 <2.34 1:100 2 3 0.59 0.75 Peanut butter 1:10 16 15 0.51 a 0.18 <2.34 <2.34 1:100 2 1 0.71 0.81 Milk chocolate 1:10 44 52 0.19 0.39 2.63 2.75 1:100 4 b 10 0.47 0.20

360 MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 Table 2. (continued) Average count, a CFU RSD r APC, log 10 CFU/g Dilution Sanita-kun Sanita-kun Sanita-kun Milk chocolate chips 1:10 2 5 0.46 0.58 <2.34 <2.34 1:100 0 1 2.24 1.05 Milk chocolate chips 1:10 2 4 0.71 0.78 <2.34 <2.34 1:100 0 0 Milk chocolate chips 1:10 3 5 0.39 0.38 <2.34 <2.34 1:100 0 0 Dry cat food 1:10 14 16 0.46 0.27 <2.34 <2.34 1:100 1 0 1.00 Dry cat food 1:10 8 6 0.31 0.50 <2.34 <2.34 1:100 0 0 Dry cat food 1:10 33 37 0.32 0.54 2.53 2.57 1:100 5 3 0.55 0.64 Frozen pizza 1:1000 406 b 285 0.02 0.09 6.61 6.46 1:10000 48 b 33 0.08 0.10 Frozen pizza 1:10000 53 a 86 0.09 0.18 6.75 6.94 1:100000 8 10 0.19 0.44 Frozen pizza 1:10000 171 b 120 0.05 0.13 6.23 6.08 1:100000 21 b 10 0.11 0.26 Frozen gyoza meat dumpling 1:10 21 13 0.20 0.55 2.32 2.15 Frozen gyoza meat dumpling Frozen gyoza meat dumpling 1:100 2 2 0.50 0.95 1:10 21 13 0.20 0.55 2.32 2.15 1:100 2 2 0.50 0.95 1:10 12 17 0.40 0.28 2.11 2.23 1:100 2 2 0.55 0.40 a Significantly higher or lower (p < 0.05). b Significantly higher or lower (p < 0.01). c The colonies in oregano by Sanita-kun were counted after 24 h incubation. lutions were applied on 5 replicate Sanita-kun sheets and incubated at 30, 32.5, 35, and 37 C for 24 48 h. Colonies were counted at 24, 27 30, 41, 44 45 and/or 48 h after incubation. (d) Limit of quantitation. Sample homogenate or dilution was prepared to contain the aerobic microorganism at ca 1 CFU/mL. Sample homogenate or dilution was inoculated to 10 replicate Sanita-kun sheets and incubated at 35 C for 48 h. Colonies on each Sanita-kun sheet were counted after incubation. (e) Lot-to-lot study. The 3 lots of Sanita-kun sheets were 9909TC (manufactured in September 1999), 00918TC (September 2000), and 001124TC (November 2000). Eleven foods were prepared as homogenates according to AOAC Official Method 966.23. Sample dilutions (1:100, 1:1000, 1:10 000) were prepared by adding sterile physiological saline. Sample homogenates and dilutions were applied on duplicate Sanita-kun sheets and incubated at 35 C for 48 h. Colonies on each Sanita-kun sheet were counted after incubation. Independent Laboratory Study Comparative Recovery (a) Sanita-kun Aerobic Count vs AOAC Official Method 966.23 Methodology. A single strain of Escherichia coli was used in the study. It was obtained from the Silliker Laboratories Research (SLR) culture collection and labeled as SLR 1392. The original source of the isolate was American Type Culture Collection (ATCC) 11229. The strain was cultivated in trypticase soy broth and incubated at 35 C for 24 h. After incubation, serial dilutions of the strain were made and analyzed for viable counts by the pour plate technique using

MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 361 Figure 3. Regression analysis of Sanita-kun sheets to plate count. trypticase soy incubated at 35 C for 24 h. The strain was held at 4 C until viable plate counts were determined. A portion of the appropriate culture dilution was inoculated into the foods to prepare the target level sample considering indigenous populations. (b). Ground beef and hot dogs, purchased from local grocery stores, were used. (c) Procedure. Each 25 g of inoculated or uninoculated sample was added to 400 ml stomacher bags; 225 ml BPB was added to each stomacher bag and samples were stomached for 2 min. Sample dilutions (1:100, 1:1000, 1:10 000) were prepared by adding BPB. Sample homogenate and dilutions were plated on 8 replicates of Sanita-kun according to package instruction and with plate count according to AOAC Official Method 966.23. (d) Statistical analyses. The difference of mean aerobic plate counts was compared by using the t-test, and the difference of standard deviation was compared with the F-ratio test. Results Internal Laboratory Study (a) Repeatability. The average counts and the relative standard deviations for repeatability (RSD r ) of 5 replicates (5 subsamples) of 4 target contamination levels (<10, 10 25, about 100, about 250 CFU/mL) of 14 categories are shown in Table 1. The contamination level, >100 CFU/mL, was not found in ice cream, raisins, and milk chocolate chips. In peanut butter, even >10 CFU/mL level contamination was not found. In all foods, the RSD r in higher contamination levels was relatively small, and large RSD r values were observed in contamination levels <10 CFU/mL. The average RSD r values of frozen or raw pork, cabbage, roast pork, frozen or raw shrimp, ice cream, fried egg, red pepper, buckwheat flour, raisins, udon noodles, peanut butter, milk chocolate chips, dry cat food, and frozen pizza were 0.17 (0.13), 0.23 (0.21), 0.15 (0.13), 0.30 (0.20), 0.17 (0.04), 0.44 (0.13), 0.31 (0.21), 0.37 (0.36), 0.3, 0.24 (0.21), 0.3, 0.33 (0.35), 0.18 (0.14), and 0.22 (0.22), respectively. (Shown in parentheses are the average RSD r values, excluding those in the contamination level <10 CFU/mL.) The average RSD r values of total foods were 0.38, 0.30, 0.19, and 0.12 in <10, 10 25, about 100, and about 250 CFU/mL target levels, respectively. The average RSD r of total foods, except those <10 average counts, was 0.19. (b) Accuracy. Most aerobic plate counts (APCs) of foods with Sanita-kun and plate count were similar (Table 2). The APCs for one lot each of frozen pork, cabbage, shelled eggs, and udon noodles and one set of 3 lots of frozen shrimp were significantly lower (p < 0.01) with Sanita-kun than with plate count, and one lot each of raw pork, frozen shrimp, and frozen pizza was significantly lower (p < 0.05). The APCs for 2 lots of frozen pork and frozen pizza were significantly higher (p < 0.05 and p < 0.01, respectively) with Sanita-kun than with plate count. The log 10 APC results of both Sanita-kun sheets and plate count were plotted on logarithmic paper. The regression analysis is shown in Figure 3. The correlation coefficient (r 2 ) of the Sanita-kun sheets compared to the plate count was calculated as 0.99. The average RSD r values of both Sanita-kun sheets and plate count on the calculated lower dilutions in each food type were 0.08 and 0.11 (frozen and raw pork), 0.13 and 0.07 (cabbage), 0.18 and 0.08 (roast pork), 0.13 and 0.13 (frozen shrimp), 0.15 and 0.16 (ice cream), 0.15 and 0.12 (shelled eggs), 0.07 and 0.13 (oregano), 0.10 and 0.14 (red pepper), 0.10 and 0.20 (buckwheat flour), 0.28 and 0.22 (raisins), 0.18 and 0.15 (udon noodles), 0.10 and 0.28 (peanut butter), 0.46 and 0.58 (milk chocolate), 0.36 and 0.44 (dry cat food), 0.05 and 0.13 (frozen pizza), and 0.28 and 0.34 (frozen gyoza). The average RSD r values of total foods were 0.18 (Sanita-kun sheets) and 0.21 (plate count ). Significant difference of the RSD r between Sanita-kun sheets and plate count was observed in some foods. The RSD r values of one lot each of cabbage and raisins were significantly higher (p < 0.01) with Sanita-kun sheets than with plate count. For one lot of frozen shrimp, the RSD r for the Sanita-kun sheets was significantly lower (p < 0.01). (c) Ruggedness. In many foods, the APC between 24 and 48 h incubation was similar (Table 3). For pork, cabbage, shrimp, and udon noodles, the APCs at 24 h incubation were lower than those at 48 h by approximately one-half. In all foods, no significant differences of APCs between 44 and 45 h and 48 h incubation at 32.5 and 35 C were observed. For raw pork, buckwheat flour, and dry cat food, no significant differences of APCs between 24 and 48 h incubation at 35 C were observed (Table 4). No significant differences were observed between 32.5 and 35 C incubation, except for cabbage, frozen shrimp, and fried egg. The APCs of cabbage, frozen shrimp, and fried egg at 32.5 C were signifi-

362 MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 Table 3. Ruggedness study (comparison of 24 and 48 h incubation) Average count, a CFU RSD r APC, log 10 CFU/g Dilution 24 h 48 h 24 h 48 h 24 h 48 h Raw pork 1:10000 291 364 0.09 0.04 6.46 a 6.57 1:100000 25 45 0.27 0.09 Cabbage 1:10000 10 47 0.15 0.18 5.00 a 5.65 1:100000 1 3 1.05 0.80 Cabbage 1:10000 90 150 0.16 0.11 5.95 a 6.18 1:100000 8 14 0.41 0.46 Frozen shrimp 1:10000 52 74 0.12 0.11 5.72 a 5.91 1:100000 6 8 0.63 0.61 Frozen shrimp 1:1000 22 42 0.19 0.04 4.32 a 4.61 1:10000 1 2 1.05 0.56 Frozen shrimp 1:1000 27 50 0.17 0.15 4.40 a 4.67 1:10000 1 2 0.91 0.71 Ice cream 1:10 31 33 0.21 0.21 2.49 2.52 1:100 3 3 0.26 0.26 Ice cream 1:10 30 32 0.16 0.17 2.49 2.52 1:100 4 5 0.34 Shelled eggs 1:10 110 157 0.16 0.13 3.00 3.15 1:100 1 2 0.96 0.71 Shelled eggs 1:10 24 33 0.22 0.19 2.36 2.52 1:100 1 3 0.91 0.62 Red pepper 1:10000 78 90 0.17 0.15 5.90 5.94 1:100000 10 12 0.36 0.29 Red pepper 1:10000 81 88 0.08 0.07 5.90 5.96 1:100000 7 8 0.41 0.40 Red pepper 1:10000 81 94 0.08 0.08 5.92 5.99 1:100000 10 14 0.24 0.15 Dry tangle 1:10 9 12 0.74 0.5 <2.34 <2.34 1:100 Dry green laver 1:10 135 193 0.05 0.05 3.18 3.32 1:100 26 41 0.14 0.15 Buckwheat flour 1:1000 61 89 0.16 0.09 4.79 b 4.95 1:10000 7 9 0.31 0.32 Buckwheat flour 1:1000 67 74 0.11 0.10 4.81 4.87 1:10000 5 8 0.27 0.11 Buckwheat flour 1:1000 46 75 0.20 0.10 4.65 4.88 1:10000 4 8 0.35 0.28 Udon noodles 1:1000 111 236 0.09 0.14 5.04 b 5.38 1:10000 9 26 0.48 0.12 Udon noodles 1:1000 19 42 0.31 0.15 3.26 3.62 1:10000 1 4 1.73 0.39 Peanut butter 1:10 10 16 0.60 0.51 <2.34 <2.34 1:100 0 1 0.96 0.71 Peanut butter 1:10 6 7 0.68 0.58 <2.34 <2.34 1:100 1 2 1.09 0.59 Milk chocolate 1:10 28 44 0.26 0.19 2.43 2.63 1:100 2 4 0.46 0.47 Dry cat food 1:10 24 33 0.25 0.32 2.41 2.53 1:100 4 5 0.54 0.55 a b Significantly low (p < 0.01). Significantly low (p < 0.05).

MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 363 Table 4. Ruggedness study (effect of incubation temperature and time) APC, log 10 CFU/g Incubation time, h Temperature, C 30 32.5 35 37 Raw pork 24 5.48 a 5.49 a 5.36 a 4.89 a 29 5.58 b 5.53 b 5.43 a 4.96 a 45 5.80 5.73 5.60 5.26 a 48 5.81 5.75 5.60 5.28 a Cabbage 24 6.20 a 6.20 a 5.85 a 5.72 a 30 6.34 a 6.30 a 6.15 a 5.76 a 48 6.63 6.62 6.53 a 6.28 a Roast pork 24 4.56 a 4.70 a 4.64 a 4.60 a 29 4.82 a 4.81 a 4.74 a 4.70 a 45 5.04 5.04 5.04 4.90 a 48 5.08 5.08 5.04 4.92 a Frozen shrimp 24 3.96 a 3.92 a 3.86 a 3.80 a 27 3.99 a 3.96 a 3.88 a 3.83 a 48 4.60 4.54 4.36 a 4.00 a Frozen shrimp 2 24 3.69 a 3.62 a 41 3.89 3.78 a 44 3.90 3.79 a 48 3.93 3.83 b Fried egg 24 3.94 c 3.56 b 3.23 a 2.95 a 29 4.00 c 3.65 3.34 a 2.98 a 45 4.04 c 3.76 3.38 a 3.11 a 48 4.08 c 3.78 3.40 a 3.11 a Red pepper 24 5.95 b 5.99 6.00 5.95 a 27 5.97 6.00 6.00 5.97 b 48 6.04 6.00 6.04 5.98 b Red pepper 2 24 5.96 5.93 a 41 6.00 5.97 44 6.04 5.99 48 6.04 6.00 Buckwheat flour 1 24 4.30 a 4.36 4.34 4.52 27 4.36 b 4.38 4.40 4.54 48 4.41 4.46 4.43 4.56 Buckwheat flour 2 24 4.32 a 4.36 a 4.40 4.40 44 4.40 4.40 4.41 4.41 48 4.40 4.41 4.43 4.43 Udon noodles 24 1.34 a 1.46 a 2.11 a 1.88 a 44 2.93 d 2.74 2.67 b 2.41 a 48 2.96 d 2.77 2.68 2.41 a Dry cat food 24 5.04 a 5.23 b 5.23 5.26 29 5.20 a 5.26 5.26 5.26 45 5.26 5.28 5.28 5.28 48 5.26 5.28 5.28 5.28 Frozen pizza 24 2.18 a 2.22 a 2.26 a 2.28 a 30 2.28 a 2.23 a 2.30 a 2.32 a 48 3.15 c 2.94 2.91 2.78 Frozen pizza 2 24 2.43 a 2.45 a 41 2.74 a 2.71 a 44 2.79 b 2.74 a 48 2.83 2.77 b a b c d Significantly lower (p < 0.01) than incubation at 32.5 C for 48 h. Significantly lower (p < 0.05) than incubation at 32.5 C for 48 h. Significantly higher (p < 0.01) than incubation at 32.5 C for 48 h. Significantly higher (p < 0.05) than incubation at 32.5 C for 48 h.

364 MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 Table 5. Ruggedness study (effect of incubation time at 32.5 C) APC, log 10 CFU/g Incubation time, h 24 27 29 30 41 44 45 48 Raw pork 5.49 a 5.53 b 5.73 5.75 Cabbage 6.20 a 6.30 a 6.66 Roast pork 4.70 a 4.81 a 5.04 5.08 Frozen shrimp 3.92 a 3.96 a 4.54 Frozen shrimp 2 3.69 a 3.89 3.90 3.93 Fried egg 3.56 a 3.65 3.76 3.78 Red pepper 5.99 6.00 6.00 Red pepper 2 5.96 6.00 6.04 6.04 Buckwheat flour 1 4.36 a 4.38 a 4.48 Buckwheat flour 2 4.36 a 4.40 4.41 Udon noodles 1.46 a 2.74 2.77 Dry cat food 5.23 b 5.26 5.28 5.28 Frozen pizza 2.22 a 2.23 a 2.94 Frozen pizza 2 2.43 a 2.74 2.79 2.83 a b Significantly lower (p < 0.01) than 48 h incubation. Significantly lower (p < 0.05) than 48 h incubation. Table 6. Ruggedness study (effect of temperature at 48 h incubation) APC, log 10 CFU/g Incubation temperature, C 30 32.5 35 37 Raw pork 5.81 5.75 5.60 5.28 a Cabbage 6.63 6.62 6.53 a 6.28 a Roast pork 5.08 5.08 5.04 4.92 a Frozen shrimp 4.60 4.54 4.36 a 4.00 a Frozen shrimp 2 3.93 3.83 b Fried egg 4.08 a 3.78 3.40 a 3.11 a Red pepper 6.04 6.00 6.04 5.98 b Red pepper 2 6.04 6.00 Buckwheat flour 1 4.41 4.46 4.43 4.56 Buckwheat flour 2 4.40 4.40 4.43 4.43 Udon noodles 2.96 a 2.77 2.68 2.41 a Dry cat food 5.26 5.28 5.28 5.28 Frozen pizza 3.15 b 2.94 2.91 2.78 Frozen pizza 2 2.83 2.77 a Significantly higher or lower than 32.5 C (p < 0.01). b Significantly higher or lower than 32.5 C (p < 0.05).

MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 365 Table 7. Limit of quantitation a Table 8. Lot-to-lot study Average count, CFU SD RSD r APC, log 10 CFU/g 9909TC 00918TC 001124TC Avg RSD r of APC Raw pork 1.6 1.4 0.88 Cabbage 2.0 1.9 0.95 Frankfurter 1.4 0.84 0.60 Raw shrimp 1.4 1.8 1.29 Ice cream 1.7 0.95 0.56 Shelled eggs 1.8 1.0 0.56 Red pepper 1.7 2.2 1.29 Buckwheat flour 1.7 1.1 0.65 Udon noodles 2.2 1.3 0.59 Peanut butter 1.7 1.3 0.76 Milk chocolate chips 1.3 0.82 0.63 Dry cat food 1.0 0.94 0.94 Frozen pizzas 1.5 1.3 0.87 Raw pork 5.91 5.89 5.93 5.91 0.05 Frankfurter 5.93 5.79 5.83 5.85 0.18 Frozen shrimp 4.96 4.80 4.86 4.87 0.20 Ice cream 2.48 2.54 2.49 2.50 0.08 Fried egg 3.49 3.46 3.49 3.48 0.03 Red pepper 5.95 5.86 5.99 5.93 0.15 Oregano 6.58 6.48 6.48 6.51 0.14 Buckwheat flour 5.15 5.15 5.18 5.16 0.04 Udon noodles 4.20 4.20 4.26 4.22 0.07 Peanut butter 2.08 2.00 2.11 2.06 0.13 Dry cat food 5.04 5.00 4.95 5.00 0.10 a SD = Standard deviation; RSD r = relative standard deviation for repeatability. cantly higher than at 35 C. The APCs at 30 C incubation were significantly higher than those at 35 C incubation except for roast pork, buckwheat flour, and dry cat food; no significant differences were seen between 30 and 32.5 C incubation except for udon noodles and fried egg. The APCs at 37 C incubation were significantly lower than those at 35 C, except for frozen pizza, buckwheat flour, and dry cat food. From these results (Tables 4 6) we can recommend incubation at 32.5 C with 2.5 C allowance, for 46 h with 2 h allowance. (d) Limit of quantitation. On nearly 1 CFU/mL, the average counts of 10 replicate Sanita-kun sheets were 1.0 2.2 and the RSD r values were 0.56 1.30 (Table 7). Lot-to-lot study. In all tested foods, APCs were similar for the 3 lots of Sanita-kun sheets. RSD r values ranged from 0.03 (fried egg) to 0.20 (frozen shrimp) as shown in Table 8. The average RSD r was calculated as 0.13. A performance decrease in the older lot of Sanita-kun sheets was not recognized and the shelf-life of the sheet was suggested to be at least 14 months. Independent Laboratory Study When the target level was <10 CFU/g, all samples were <10 CFU/g by Sanita-kun sheets, and all samples except one were <10 CFU/g by the AOAC Official Method. In ground beef, no significant difference was observed among the APCs of the AOAC Method and 20 and 48 h incubation of Sanita-kun sheets. In the target levels of 100 250 and 1000 CFU/g hot dogs, the APCs of Sanita-kun sheets were significantly higher (p < 0.05) Table 9. Independent validation study AOAC Method Sanita-kun, 20 ±2h Sanita-kun, 48 h Target level, CFU/g Average APC, CFU/g RSD r Average APC, CFU/g RSD r Average APC, CFU/g RSD r Hot dogs <10 <10 except 1 replicate <10 <10 100 250 400 0.14 510 a 0.16 500 a 0.14 1000 1100 0.14 1300 a 0.11 1300 a 0.11 2500 4100 0.17 4400 0.14 4500 0.14 Ground beef <10 <10 <10 <10 100 250 410 0.13 380 0.16 400 0.15 1000 920 0.26 780 0.31 820 0.24 2500 3500 0.12 3900 0.16 4200 0.19 a Significantly higher (p < 0.05) than AOAC Method.

366 MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 than those of the AOAC Method (Table 9). In the average APC and RSD r of each target level of Sanita-kun sheets, no significant difference was observed between 20 and 48 h incubation. The analyst pointed out that the Sanita-kun sheets were easy to use and read. Discussion We did not detect aerobic microorganisms in food dyes (data not shown). dyes interrupted the color visualization of the colonies on the Sanita-kun sheets. Therefore, we withdrew food dyes from the claims of the food matrixes. In the repeatability study, the average RSD r of fried egg was higher (0.57). This was caused by the low actual mean counts (1 and 2) in the lower 2 target levels (<10, 10 25). In the accuracy study, the higher average RSD r values of milk chocolate and dry cat food by the Sanita-kun and plate count method were caused by low counts. The APCs of several lots were significantly higher or lower (p < 0.05 or p < 0.01) than those of the plate count. However, the difference was small; therefore, we believe there will be practically no problem in daily monitoring of such foods. In the independent validation study, the analyst pointed out that the Sanita-kun sheets were easy to use and read. She also pointed out, however, that the colony was sometimes more diffuse, with string-like spreading at 48 h; therefore, colonies that had been counted as 2 at 20 h may have been counted as one at 48 h. However, she reported quite similar counts between 20 and 48 h (Table 9). In the independent validation study, BPB was used to prepare the homogenate and sample dilutions. We often observed spread colonies on the Sanita-kun sheets after 48 h incubation when buffers or solutions without 0.5 0.9% NaCl were used for sample preparation and dilution. Spread colonies were not observed on the sheets when buffers or solutions containing 0.5 0.9% NaCl were used. We recognized increases in colonies on the Sanita-kun sheets at 48 h from 24 h incubation in several types of foods (Tables 3 6). To obtain precise numbers of colonies on the Sanita-kun sheets, 48 h incubation is preferable. From results of further ruggedness study, we recommend incubation at 32.5 2.5 C for 46 2 h. The 24 h incubation is enough in a daily monitoring of food contamination except for udon noodles, because the differences of log 10 counts between 24 and 48 h incubation at 32.5 C was within 1. Sanita-kun Aerobic Counts sheets contain TTC as growth indicator; the Petri-film AC plate also uses the same indicator. The performance of Sanita-kun Aerobic Counts and the Petri-film AC plate was compared on about 300 foods (data not shown). The APCs of both methods were similar and the correlation coefficient (r 2 ) was calculated as 0.99. At times, degradation of the Petri-film AC plate and liquefaction of the gel by bacteria and diffused colonies were observed. The degradation of water-soluble polymer or the nonwoven fabric in the Sanita-kun sheets was never observed. Sanita-kun sheets are not required for laborious medium preparation, and the sheets have the advantages of being space saving and causing small waste when compared with conventional medium. The Sanita-kun aerobic count was similar to that with plate count. We believe that the Sanita-kun sheets provide an alternative method to plate count. The Sanita-kun Aerobic Counts has been granted AOAC Performance Tested Method SM status. Acknowledgements We thank Wendy Lepper and Ann M. Schulz (Silliker Laboratory Corporate Research Center) for their extensive works in independent laboratory study.