Current methods for enumeration of total aerobic microorganisms

FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 257 FOOD BIOLOGICAL CONTAMINANTS Enumertion of Totl Aerobic Microorgnisms in Foods by SimPlte Totl Plte Count Color Indictor Methods nd Conventionl Culture Methods: Collbortive Study PHILIP T. FELDSINE, STEPHANIE C. LEUNG, ANDREW H. LIENAU, nd LINDA A. MUI BioControl Systems Inc., 12822 SE 32nd St, Bellevue, WA 98005 DAVID E. TOWNSEND IDEXX Lbortories Inc., One IDEXX Dr, Westbrook, ME 04092 Collbortors: V. Arling; L. August; D. Brhm; M. Bohnert; A. Boville; S. Brookmn; C. Chvey; S. Clements; R. Dvis; S. Devne; S. Dissel; S. Grtside; D. Hgel; C. Hernndez; S. Hopkins; M. Howell; F. Humbert; J. Hunsucker; J. Jckson; S. Koch; C. Kuber; J. Lmb; L. Lewis; B. Lightfoot; W. Lin; S. Musch; K. Nieves; M. Poumeyrol; S. Qvist; J. Rice; D. Solis; J. Terry; P. in t Veld; R. Voermns; D. Wrburton; J. Welch The reltive efficcy of the SimPlte Totl Plte Count Color Indictor (TPC CI) method (SimPlte ) ws compred with the Officil Method 966.23 ( ) for enumertion of totl erobic microorgnisms in foods. The SimPlte TPC CI method, incubted t (SimPlte ), ws lso compred with the Interntionl Orgniztion for Stndrdiztion () 4833 method ( ). Six food types were nlyzed: ground blck pepper, flour, nut mets, frozen hmburger ptties, frozen fruits, nd fresh vegetbles. All foods tested were nturlly contminted. Nineteen lbortories throughout North Americ nd Europe prticipted in the study. Three method comprisons were conducted. In generl, there ws <0.3 men log count difference in recovery mong the SimPlte methods nd their corresponding reference methods. Men log counts between the 2 reference methods were lso very similr. Repetbility (s r ) nd reproducibility (s R ) stndrd devitions were similr mong the 3 method comprisons. The SimPlte method (35 C) nd the method were comprble for enumerting totl erobic microorgnisms in foods. Similrly, the SimPlte method () ws comprble to the method when smples were prepred nd incubted ccording to the method. Submitted for publiction November 2002. The recommendtion ws pproved by the Methods Committee on Microbiology nd Extrneous Mterils s First Action. See Officil Methods Progrm Actions, (2003) Inside Lbortory Mngement, Jnury/Februry issue. Corresponding uthor s e-mil: ptf@biocontrolsys.com. Current methods for enumertion of totl erobic microorgnisms include the culture method (1) nd the Interntionl Orgniztion for Stndrdiztion () method (2). Both methods use pour pltes tht re incubted for 48 h () to 72 h () before obtining results. The SimPlte Totl Plte Count Color Indictor (TPC CI) uses binry detection technology (BDT) to enumerte totl erobic microorgnisms in foods fter only 24 28 h of incubtion, thereby offering significnt timesving dvntge over the current culture methods. The SimPlte device lso elimintes uncertinty in enumertion of plte counts cused by overcrowding, spreding colony types, nd food prticulte interference on conventionl pour plte methods. In the SimPlte multiple test formt, prepred food test portions re plced onto the center of SimPlte device nd TPC CI liquid medium is dded. For the single test formt, premixed test portion/medium homogente is dispensed into the test device. The test portion/medium homogente is distributed into fixed number of individul incubting wells: 84 for norml counting rnge SimPlte nd 198 for the high counting rnge. With SimPlte, foodborne microorgnisms re suspended in nutritionlly defined growth medium. Discrete liquots re seprtely comprtmentlized nd isolted from ech other in the incubting wells where biochemicl ctivities of vible microorgnisms re monitored in liquid environment. Detection by this biochemicl process requires fewer microorgnisms to produce detectble signl in SimPlte well thn the number required to form clerly visible colony in n gr plte. Enumertion is mesured by simple binry rection; ech well is either positive or negtive. Any color chnge from the originl bckground color in ech well or in the sponge is interpreted s positive rection. Aerobic microorgnisms re enumerted by counting the numbers of wells in ech plte tht exhibit color chnge (positive wells) fter incubtion. The finl count per plte is derived from Tble 1, which is bsed upon the principle of the Poisson distribution. The lrge

258 FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 Tble 1. SimPlte conversion tble Wells = popultion 1=2 31=76 61=216 2=4 32=80 62=224 3=6 33=84 63=232 4=8 34=86 64=240 5=10 35=90 65=248 6=12 36=94 66=256 7=14 37=96 67=266 8=16 38=100 68=276 9=18 39=104 69=288 10 = 22 40 = 108 70 = 298 11 = 24 41 = 112 71 = 312 12 = 26 42 = 116 72 = 324 13 = 28 43 = 120 73 = 338 14 = 30 44 = 124 74 = 354 15 = 32 45 = 128 75 = 372 16 = 36 46 = 132 76 = 392 17 = 38 47 = 136 77 = 414 18 = 40 48 = 142 78 = 440 19 = 42 49 = 146 79 = 470 20 = 46 50 = 150 80 = 508 21 = 48 51 = 156 81 = 556 22 = 50 52 = 160 82 = 624 23 = 54 53 = 166 83 = 738 24 = 56 54 = 172 84 = >738 25 = 58 55 = 178 If there re no positive 26=62 56=184 wells, nd the sponge is positive, 27=64 57=190 popultion is 1 28 = 68 58 = 196 If there re no positive 29=70 59=202 wells, nd the sponge is negtive, 30=74 60=208 popultion is <1 The popultion reflects the number of microorgnisms per plte. To determine the number of microorgnisms per g (ml) food product, refer to I(c), Reding nd Interprettion of Results. number of wells llows ccurte mesure of the true microbil popultion in test smple. A recent methods comprison study compred the SimPlte TPC CI method nd the method for enumertion of totl erobic microorgnisms in 20 different foods (unpublished). Overll, the SimPlte method produced equivlent or sttisticlly greter men log counts for ll food types nlyzed, except for one lot of fresh fruit nd one lot of fresh juice. The results indicted tht SimPlte TPC CI is equivlent to the pour plte reference method. A seprte interntionl multilbortory collbortive study compred the reltive effectiveness of the SimPlte TPC CI method with tht of the pour plte method for recovery of totl erobic microorgnisms. The SimPlte method nd the pour plte method were lso evluted in this sme study. Collbortive Study Study Design Six food types representtive of wide rnge of food ctegories were evluted: ground blck pepper, frozen fruits, flour, nut mets, frozen hmburger ptties, nd fresh vegetbles. Two test portions from 3 different lots were nlyzed for ech food type (totl of 6 test portions per food type). All foods tested were nturlly contminted. Collbortors were sent 2 sets of rndomized test portions; one set ws used for nlysis by the SimPlte method incubted t (SimPlte 35) nd the pour plte method incubted t ( 35). The other set ws used for nlysis by the SimPlte method incubted t (SimPlte 30) nd the pour plte method incubted t ( 30). The blck pepper, flour, nd nut mets were stored t room temperture until the dy of nlysis. Fresh vegetbles were kept refrigerted until nlysis. Frozen fruit nd frozen hmburger ptty test portions were kept frozen ( 20 C) nd tested unthwed. For one set of test portions, collbortors were instructed to prepre the initil suspension nd further deciml dilutions s recommended by Officil Method 966.23 (1). The pproprite dilutions were nlyzed by the pour plte method ( 966.23) nd the SimPlte method, both incubted t. For the second set of test portions, collbortors prepred the initil suspension nd further deciml dilutions ccording to 6887 (3). The pproprite dilutions were nlyzed ccording to 4833 (2) nd the SimPlte method, both incubted t. Results obtined were submitted on summry forms with the pproprite rw dt. A minimum of 8 lbortories submitted vlid dt for ech food type. Test Portion Preprtion The nturlly contminted foods were purchsed t the retil level in Settle, WA. The bulk foods were thoroughly mixed nd then pckged into 50 g test portions for shipment to collbortors. Test Portion Distribution Ech collbortor received 2 sets of 6 test portions for ech week the study ws conducted. Test products were distributed by overnight delivery from the United Sttes to lbortories throughout North Americ nd Europe. Frozen foods were shipped on dry ice; ground blck pepper, flour, nd nut met test portions were shipped t room temperture. Fresh vegetbles were shipped on ice to North Americn collbortors only. Test Portion Anlysis The nd culture methods use different diluents for initil suspension nd deciml dilutions of test portions. This required collbortors to set up seprte pired smples

FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 259 with one test portion prepred with the diluent nd the other test portion prepred with the diluent. Designted food test portions were prepred s specified by Method 966.23. For ll food types, except nut mets, 50 g test portions were dded to 450 ml Butterfield s phosphte buffered diluent (BPBD) wter nd homogenized by blending for 2 min. For nut mets, 50 g test portions were dded to 50 ml BPBD nd vigorously shken to produce the 10 0 dilution. After homogeniztion, test portions were serilly diluted into BPBD. The number of dilutions necessry to perform the test depended on the food type being nlyzed. Subsequent 10-fold dilutions were prepred by dding 10 ml of the previous dilution to 90 ml sterile diluent nd shken 25 times in 30 cm rc. BioControl provided the suggested dilution series for ech food type the week before nlysis ws initited. From ech pproprite dilution, 1 ml liquot ws trnsferred to sterile Petri dish in duplicte. A 12 15 ml mount of Stndrd Methods Agr (SMA) tempered to 42 45 C ws dded nd swirled to evenly distribute the dilution nd gr. Plte counts of ech dilution level were performed in duplicte. All pltes were incubted t for 48 2 h. All pltes were counted fter the required incubtion. Dt were recorded onto pproprite worksheets. For the method, pltes contining 30 300 colonies were counted. Designted food test portions were lso prepred s specified by method 6887. For ll food types, 50 g test portions were dded to 450 ml peptone slt solution nd homogenized by mcerting for 1 min. After homogeniztion, test portions were serilly diluted into peptone slt solution. The number of dilutions necessry to perform the test depended on the food type being nlyzed. Subsequent 10-fold dilutions were prepred by dding 10 ml of the previous dilution to 90 ml sterile diluent nd mixed well. BioControl provided the suggested dilution series for ech food type the week before nlysis ws initited. From ech pproprite dilution, 1 ml liquot ws trnsferred to sterile Petri dish in duplicte. A 15 ml mount of SMA tempered to 44.5 45.5 C ws dded nd swirled to evenly distribute the diluted food test portion nd gr. Plte counts of ech dilution level were performed in duplicte. All pltes were incubted t 30 1 C for 72 3 h. All pltes were counted fter the required incubtion. Dt were recorded onto pproprite worksheets. Diluted test portions prepred for the nd reference methods were lso used for SimPlte nlyses. One set of SimPltes ws set up using the dilutions from the method. A seprte set of SimPltes ws set up using the dilutions from the method.a 1 ml mount of the diluted test portion ws trnsferred to the center of the SimPlte device. The SimPlte ws overlyed with 9 ml rehydrted TPC CI medium, for finl volume of 10 ± 0.2 ml. After the plte ws covered with the lid, the food/medium homogente ws mixed nd swirled into the wells. Excess liquid ws decnted into the integrted collection sponge. The SimPlte devices from the test portions were incubted upright t 35 1 C for 24 28 h. The SimPlte devices from the test portions were incubted upright t 30 1 C for 24 28 h. After incubtion, the number of wells with color chnge from the originl bckground color ws counted. Tble 1 ws used to correlte the number of positive wells to the totl erobic microorgnisms per SimPlte device by mtching the number of positive wells counted to the corresponding microbil popultion given in Tble 1. If dilution ws mde to the originl food test portion before inocultion of the SimPlte device, then the totl erobic microorgnisms per grm of food ws clculted by multiplying the popultion from Tble 1 by the dilution fctor. Sttisticl Anlysis For ech lot of food, duplicte plte counts were verged nd reported s erobic plte count per grm (APC/g) for the or reference methods, nd the SimPlte methods. The bse 10 logrithms of SimPlte counts nd APC/g for ech of the reference culture methods were used for sttisticl nlysis. Repetbility (s r ) nd reproducibility (s R ) stndrd devitions, reltive stndrd devitions of repetbility (RSD r ) nd reproducibility (RSD R ), nd repetbility (r) nd reproducibility (R) vlues were clculted ccording to the methods of Youden nd Steiner (4) fter outliers were eliminted. The Cochrn test ws used to remove lbortories showing significntly greter vribility mong replicte nlyses thn other lbortories for prticulr set of test portions. An F sttistic tht computes the rtio of the 2 vrinces ws used to compre repetbility nd reproducibility vrinces. Men responses between the 2 methods were compred by using 2-smple (pired) t-test. Officil Method 2002.07 Detection nd Quntifiction of Totl Aerobic Microorgnisms SimPlte Totl Plte Count Color Indictor (TPC CI) Method First Action 2002 (Applicble to detection nd quntifiction of totl erobic microorgnism popultions in milk chocolte, cke mix, ground pepper, nut mets, diry foods, red mets, poultry mets, sefoods, lunch met, frozen pot pies, cerels, pst, egg products, flour, hsh brown pottoes, vegetbles, fruits, nd fruit juice.) Cution: Test portion dilutions nd incubted SimPlte devices from food products could contin pthogenic bcteri if the prticulr test portion ws contminted. Stndrd septic microbiologicl lbortory techniques, including decontmintion of ny spills with disinfectnt, re recommended. See Tbles 2002.07A C for the results of the interlbortory study supporting cceptnce of the method. A. Principle SimPlte Totl Plte Count Color Indictor (TPC CI) method is used for detection nd quntifiction of totl erobic popultions. The TPC CI medium nd food mixture is dispensed into SimPlte device nd incubted for 24 28 h. The

260 FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 Tble 2002.07A. Sttisticl nlysis of interlbortory results for totl erobic microorgnisms by the 35 nd SimPlte 35 methods Men b s r e RSD r, % f r g s R h RSD R, % i R j Food group Lot N c SimPlte d c SimPlte d c SimPlte d c SimPlte d c SimPlte d c SimPlte d c SimPlte d Pepper A 12 6.04 6.13 0.09 k 0.15 1.50 2.50 0.25 0.43 0.22 0.16 l 3.70 2.60 0.63 0.44 B 13 7.30 7.53 k 0.11 0.14 1.60 1.80 0.32 0.38 0.35 0.17 k 4.80 2.30 0.99 0.48 C 14 6.44 6.66 k 0.11 l 0.16 1.70 2.30 0.31 0.44 0.34 0.22 l 5.30 3.30 0.96 0.61 Flour Rye 14 5.73 5.83 0.15 0.18 2.70 3.10 0.43 0.50 0.27 0.28 4.70 4.70 0.76 0.77 Whet 11 3.87 3.93 0.13 l 0.19 3.30 4.80 0.35 0.53 0.30 l 0.46 7.80 11.80 0.85 1.30 White 13 4.13 4.24 0.34 0.27 8.30 6.40 0.96 0.76 0.36 0.45 8.70 10.50 1.00 1.25 Nut mets Penuts 13 4.04 4.24 l 0.29 l 0.41 7.30 9.70 0.83 1.15 0.33 0.37 8.20 8.60 0.92 1.02 Almonds 13 2.42 2.53 0.11 k 0.23 4.70 9.10 0.32 0.64 0.31 0.29 12.90 11.60 0.88 0.82 Hzelnuts 12 3.67 3.72 0.69 0.57 18.80 15.30 1.93 1.59 0.65 0.69 17.60 18.60 1.81 1.93 Frozen ptties A 15 2.78 3.01 0.13 k 0.25 4.30 7.90 0.35 0.69 0.27 l 0.38 9.40 12.10 0.76 1.06 B 15 3.84 4.06 0.31 0.17 k 8.20 4.10 0.88 0.47 0.53 0.50 13.90 12.40 1.49 1.41 C 15 2.85 2.86 0.14 l 0.20 5.00 7.01 0.40 0.57 0.26 l 0.35 8.90 12.40 0.71 0.99 Frozen fruits Peches 10 3.24 3.37 0.14 0.14 4.30 4.30 0.39 0.40 0.27 0.33 8.20 9.70 0.74 0.92 Strwberries 12 3.96 4.33 k 0.14 0.12 3.60 2.60 0.40 0.32 0.27 0.21 6.80 4.90 0.75 0.60 Blueberries 10 2.85 3.08 k 0.17 0.15 5.90 4.80 0.47 0.42 0.20 0.28 7.10 9.10 0.57 0.78 Fresh vegetbles Crrots 12 7.27 7.30 0.27 0.31 3.70 4.20 0.75 0.86 0.58 0.47 7.90 6.40 1.61 1.31 Broccoli 12 7.20 7.22 0.26 0.18 l 3.60 2.50 0.73 0.50 0.61 0.42 l 8.50 5.90 1.71 1.19 Celery 10 7.47 7.40 0.13 0.19 1.70 2.50 0.36 0.53 0.74 0.36 k 9.90 4.90 2.07 1.01 Number of lbortories with vlid dt. Men log erobic microorgnisms/g. Stndrd Methods Agr incubted t. SimPlte method incubted t. Repetbility stndrd devition. Repetbility reltive stndrd devition. Repetbility vlues, 2.8 s r. Reproducibility stndrd devition. Reproducibility reltive stndrd devition. Reproducibility vlues, 2.8 s R. Significntly different t p < 0.01. Significntly different t p < 0.05. b c d e f g h i j k l

FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 261 Tble 2002.07B. Sttisticl nlysis of interlbortory results for totl erobic microorgnisms by the 30 nd SimPlte 30 methods Men b s r e RSD r, % f r g s R h RSD R, % i R j Food group Lot N c SimPlte d c SimPlte d c SimPlte d c SimPlte d c SimPlte d c SimPlte d c SimPlte d Pepper A 14 5.69 5.71 0.13 0.14 2.20 2.40 0.35 0.39 0.41 0.41 7.30 7.20 1.16 1.14 B 14 7.20 7.29 0.17 k 0.35 2.40 4.80 0.48 0.98 0.34 l 0.51 4.70 7.00 0.95 1.42 C 14 6.33 6.36 0.14 l 0.21 2.20 3.20 0.39 0.58 0.39 0.35 6.20 5.50 1.10 0.97 Flour Rye 14 5.74 5.84 0.22 0.24 3.90 4.00 0.62 0.66 0.41 0.30 l 7.20 5.10 1.16 0.84 Whet 14 3.86 3.79 0.13 k 0.23 3.30 6.00 0.36 0.64 0.35 0.33 9.10 8.80 0.98 0.93 White 14 4.33 4.46 0.12 0.13 2.70 2.80 0.33 0.35 0.27 0.24 6.30 5.40 0.77 0.67 Nut mets Penuts 12 4.31 4.28 0.41 0.41 9.60 9.60 1.15 1.15 0.67 0.67 15.50 15.70 1.86 1.89 Almonds 12 2.29 2.52 0.16 l 0.23 7.00 9.10 0.45 0.65 0.57 0.43 25.00 17.10 1.60 1.21 Hzelnuts 11 3.35 3.40 0.77 0.83 23.10 24.50 2.17 2.33 0.86 0.89 25.50 26.20 2.40 2.49 Frozen ptties A 14 3.16 3.13 0.13 0.14 4.00 4.40 0.36 0.39 0.23 0.23 7.30 7.30 0.65 0.64 B 13 5.18 5.11 0.06 k 0.10 1.10 1.90 0.17 0.27 0.23 k 0.44 4.50 8.60 0.66 1.24 C 15 3.19 3.06 0.06 k 0.14 2.00 4.50 0.18 0.39 0.59 0.43 17.60 14.10 1.58 1.21 Frozen fruits Peches 13 3.70 3.92 0.11 k 0.21 2.90 5.30 0.30 0.59 0.47 0.32 l 12.80 8.10 1.32 0.89 Strwberries 11 3.95 4.13 l 0.09 l 0.14 2.30 3.40 0.26 0.40 0.35 0.16 k 9.00 3.90 0.99 0.45 Blueberries 13 2.66 2.78 0.16 0.20 6.10 7.20 0.46 0.56 0.25 0.32 9.50 11.50 0.71 0.90 Fresh vegetbles Crrots 11 7.49 7.56 0.30 0.38 3.90 5.00 0.83 1.06 0.58 0.63 7.70 8.30 1.63 1.76 Broccoli 13 7.80 7.89 0.29 0.24 3.70 3.00 0.81 0.66 0.53 0.58 6.80 7.30 1.49 1.62 Celery 11 8.58 8.49 0.08 k 0.43 0.90 5.00 0.23 1.20 0.52 0.73 6.10 8.60 1.46 2.04 Number of lbortories with vlid dt. Men log erobic microorgnisms/g. Stndrd Methods Agr incubted t. SimPlte method incubted t. Repetbility stndrd devition. Repetbility reltive stndrd devition. Repetbility vlues, 2.8 s r. Reproducibility stndrd devition. Reproducibility reltive stndrd devition. Reproducibility vlues, 2.8 s R. Significntly different t p < 0.01. Significntly different t p < 0.05. b c d e f g h i j k l

262 FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 Tble 2002.07C. Sttisticl nlysis of interlbortory results for totl erobic microorgnisms by the 30 nd 35 methods Men b s r e RSD r, % f r g s R h RSD R, % i R j Food group Lot N c d c d c d c d c d c d c d Pepper A 14 5.71 5.91 0.10 0.09 1.70 1.50 0.28 0.24 0.40 0.46 7.00 7.70 1.12 1.28 B 13 7.20 7.30 0.18 0.11 k 2.50 1.60 0.50 0.32 0.35 0.36 4.90 4.90 0.99 0.99 C 14 6.30 6.44 0.14 0.11 2.20 1.70 0.39 0.31 0.39 0.34 6.20 5.30 1.10 0.95 Flour Rye 13 5.69 5.73 0.12 0.16 2.20 2.70 0.34 0.44 0.36 0.28 6.40 4.70 1.02 0.79 Whet 10 3.75 3.83 0.16 0.11 4.20 2.80 0.44 0.31 0.26 0.32 7.00 8.40 0.74 0.91 White 12 4.33 k 4.13 0.08 l 0.35 1.80 8.50 0.21 0.98 0.27 0.37 6.20 9.00 0.75 1.04 Nut mets Penuts 12 4.31 4.05 0.41 0.30 9.60 7.40 1.15 0.84 0.67 0.34 l 15.50 8.40 1.86 0.95 Almonds 12 2.29 2.32 0.16 0.10 k 7.00 4.20 0.45 0.29 0.57 0.32 l 25.00 13.00 1.60 0.89 Hzelnuts 12 3.34 3.62 0.78 1.18 23.30 33.70 2.18 3.29 0.88 1.20 26.20 34.60 2.45 3.37 Frozen ptties A 14 3.19 l 2.93 0.14 0.14 4.30 4.60 0.39 0.38 0.23 0.28 7.30 9.50 0.65 0.78 B 14 5.14 l 3.79 0.19 l 0.32 3.70 8.50 0.53 0.90 0.33 k 0.52 6.40 13.60 0.92 1.44 C 15 3.20 l 2.86 0.07 l 0.14 2.00 5.00 0.18 0.40 0.57 0.26 l 17.90 9.00 1.60 0.72 Frozen fruits Peches 11 3.86 l 3.25 0.09 l 0.17 2.40 5.40 0.26 0.49 0.34 0.27 8.90 8.20 0.96 0.74 Strwberries 11 3.95 3.97 0.09 k 0.14 2.30 3.50 0.25 0.39 0.35 0.28 8.90 7.00 0.99 0.77 Blueberries 10 2.71 2.85 k 0.15 0.17 5.60 5.90 0.43 0.47 0.20 0.20 7.30 7.10 0.55 0.57 Fresh vegetbles Crrot 11 7.49 7.33 0.30 0.28 3.90 3.70 0.83 0.76 0.58 0.55 7.70 7.50 1.62 1.54 Broccoli 11 7.82 l 7.15 0.29 0.26 3.70 3.60 0.81 0.73 0.39 k 0.57 5.00 8.00 1.09 1.60 Celery 9 8.69 l 7.39 0.08 k 0.12 0.90 1.60 0.23 0.34 0.42 k 0.74 4.90 10.10 18.00 2.08 b c d e g h k Number of lbortories with vlid dt. Men log erobic microorgnisms/g. Stndrd Methods Agr incubted t. Stndrd Methods Agr incubted t. Repetbility stndrd devition. Repetbility reltive stndrd devition. Repetbility vlues, 2.8 s r. Reproducibility stndrd devition. Reproducibility reltive stndrd devition. Reproducibility vlues, 2.8 s R. Significntly different t p < 0.05. Significntly different t p < 0.01. f i j l

FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 263 medium chnges color in the presence of erobic microorgnisms. The totl erobic microorgnisms count is determined by counting the wells with chnged color nd correlting the number of positive wells with the number of totl erobic microorgnisms found in Tble 1. B. Medi nd Regents () Dehydrted Totl Plte Count Color Indictor (TPC CI) medium. In individully pckged single or multiple test formt. (b) Supplement A (optionl). Add 1.0 ml sterile Supplement A solution per 100 ml sterile deionized H 2 O. Alterntively, dd 1.0 ml Supplement A to 100 ml deionized H 2 O nd utoclve for 15 min t 121 C. (c) Butterfield s phosphte buffered diluent (BPBD). See 966.23(m). (d) Peptone slt solution. Dissolve 1.0 g enzymtic digest of csein nd 8.5 g NCl in 1 L deionized H 2 O. Autoclve for 15 min t 121 C. Finl ph, 7.0 0.2 t 25 C. (e) SimPlte devices. Twenty devices per pckge. Items (), (b), nd (e) re vilble from BioControl Systems, Inc. (12822 SE 32nd St, Bellevue, WA 98005). C. Apprtus () Incubtor. Mintining 35 37 C. (b) Micropipetor. Accurtely dispensing 0.1 nd 1.0 ml. (c) Pipets. Accurtely dispensing 1.0 nd 10 ml. (d) Blender/stomcher. Wring, or equivlent, for blending test portions; IUL Instruments (Cincinnti, OH) mstictor, or equivlent, for mcerting test portions. Note: A blender is used if testing in ccordnce with the method; stomcher is used if testing in ccordnce with the method. D. Generl Instructions Do not use expired medi. Store reconstituted medium between 15 nd 25 C in the drk nd use within 12 h. Dispose of medium in decontmintion continer, nd sterilize before discrding. E. Test Portions Preprtion () Weigh 50 g test portion into 450 ml sterile diluent, e.g., BPBD ( method) or peptone slt solution ( method). This is 1:10 dilution. Mcerte or blend to homogenize. (b) If lterntive test portion size is specified in testing procedure, prepre 10% (w/v) suspension. (c) If necessry, prepre 10-fold seril dilutions pproprite for the nticipted popultion of the test portion. F. TPC CI Test Procedure, Single Test Medium () For 1.0 ml test portion size. Resuspend powdered medium with 9.0 ml sterile deionized wter contining 1 ml Supplement A per 100 ml wter. Add 1.0 ml prepred test portion nd mix well. Do not count this reconstitution s dilution. (b) For 0.1 ml test portion size. Resuspend powdered medium with 9.9 ml sterile deionized wter contining 1 ml Supplement A per 100 ml wter. Add 0.1 ml prepred test portion nd mix well. This is n dditionl 1:10 dilution from E. Note: The finl volume of test portion/medium mixture in the continer should be 10 0.2 ml. (c) Remove lid from SimPlte device nd trnsfer test portion/medium mixture onto center of plte. Immeditely replce lid. Continue with H(). G. TPC CI Test Procedure, Multiple Test Medium () Empty contents of one continer into 100 ml sterile deionized wter contining 1 ml Supplement A per 100 ml wter. Shke to completely dissolve. (b) Remove lid from SimPlte device. Pipet prepred test portion onto center of plte. If prepred test portion size is 1.0 ml, overly test portion with 9.0 ml medium. Do not count this medi ddition s dilution. (c) For 0.1 ml prepred test portion, overly with 9.9 ml medium; this is n dditionl 1:10 dilution of test portion from E. Note: The finl volume of test portion/medium mixture on the plte should be 10 0.2 ml. (d) Immeditely replce the lid. Continue with H(). H. Test Procedure for Single nd Multiple Tests () Gently swirl to distribute test portion/medium mixture into ll wells. Hold plte with both hnds, tilted slightly to help distribute medium into wells. (b) Pour off excess medium by holding lid ginst plte on either side of sponge cvity. Tip plte towrd you to llow liquid to drin into sponge. Observe bckground color of wells. Bckground is defined s color of the test portion/medium mixture inside the wells before incubtion. (c) If testing in ccordnce with /BAM/USDA methods, incubte SimPlte devices in n upright position in the drk for 24 28 h t 35 ±1 C(32±1 C for diry products). If testing in ccordnce with procedures, incubte devices in n upright position in the drk for 24 28 h t 30 ± 1 C. I. Reding nd Interprettion of Results () After incubtion, observe color chnge of liquid in wells. Disregrd prticulte mtter if present. Count number of wells showing color chnge from the bckground color. The most common color chnge produced by microorgnisms is pink, but ornge, pech, red, brown, nd white my lso be observed. (b) To determine the popultion, perform the following clcultions: (1) Count the number of positive wells on the plte; (2) use Tble 1 to determine totl number of microorgnisms per plte. (c) To clculte number of microorgnisms per grm (ml), multiply the count in I(b)(2) by the pproprite dilution fctor (see E nd F for Single test, or E nd G for Multiple test). Ref.: J. Int. 86, 259 263(2003) Results Four methods for enumerting totl erobic microorgnisms in foods were compred in this collbortive study:

264 FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 pour plte method incubted t ( 35), pour plte incubted t ( 30), SimPlte method incubted t (SimPlte 30), nd SimPlte method incubted t (SimPlte 35). Nineteen lbortories throughout North Americ nd Europe prticipted in the study (Tble 2). Eight lbortories nlyzed ll 6 food types, 6 lbortories nlyzed 5 food types, one lbortory nlyzed 4 food types, nd 4 lbortories nlyzed one food type (Tble 2). The plte counts for individul test portions re presented in Tbles 3 8. Repetbility nd reproducibility nlyses re presented in Tbles 2002.07A C. Ground Blck Pepper Three lots of ground blck pepper (Lots A C) were nlyzed (Tble 3). Throughout the nlysis of ground blck pepper, some lbortories reported erobic plte counts for certin test portions tht were not in the suitble counting rnge for or methods (Tble 3). Dt from these pired test portions were not used for sttisticl nlysis. Dt generted by the 35 nd the SimPlte 35 methods were compred sttisticlly. Lbortory 7 ws determined to be n outlier for Lot A by the Cochrn test; therefore, the pired dt were excluded from sttisticl nlysis. Overll, there were 12, 13, nd 14 lbortories tht submitted vlid dt for Lots A C, respectively. Men log counts recovered from Lot A were similr for the 2 methods. Lots B nd C contined higher men log counts s mesured by the SimPlte method (p < 0.01). The repetbility (s r ) nd reproducibility (s R ) stndrd devitions (SDs) of the 2 methods were sttisticlly nlyzed for the 3 lots (Tble 2002.07A). For Lots A nd C, the method reported better SD r vlues. However, the SimPlte method showed better SD R vlues for Lots A C. Dt generted by the 30 nd SimPlte 30 methods were compred sttisticlly. Overll, 14 lbortories submitted vlid dt for the 3 lots of pepper nlyzed. The men log counts for the 30 nd the SimPlte 30 methods were not significntly different for the 3 lots nlyzed. The method reported better SD r vlues for Lots B nd C test portions. The method lso reported better SD R vlues for Lot B (p < 0.01). The SD R vlues were not significntly different for Lots A nd C between the 2 methods (Tble 2002.07B). Dt generted by the 30 nd the 35 methods were compred sttisticlly. Overll, 14 lbortories submitted vlid dt for Lots A nd C, nd 13 lbortories submitted vlid dt for Lot B. The men log counts from the 35 nd the 30 methods were not significntly different for Tble 2. Collbortor prticiption for SimPlte TPC CI interlbortory study by food type Lb Ground blck pepper Flour Nut mets Frozen hmburger ptties Frozen fruits Fresh vegetbles 1 Y Y Y Y Y Y 2 Y Y b Y Y Y Y 3 Y Y Y Y Y Y 4 Y Y Y Y Y Y 5 Y Y Y Y Y Y 6 N Y Y Y Y Y 7 Y Y Y Y Y Y 8 Y Y Y Y Y b Y 9 Y Y Y Y Y N 10 Y Y Y Y Y N 11 Y Y Y Y Y N 12 Y Y N Y Y N 13 Y Y Y b Y Y N 14 Y Y Y Y Y N 15 Y Y Y Y Y Y 16 N N N N N Y 17 N N N N N Y 18 N N N N N Y 19 N N N N N Y Totl c 14 15 14 15 15 13 b c Y = Collbortor nlyzed this food type; N = collbortor did not nlyze this food type. Lbortory did not follow study instructions. Results were not included in the sttisticl nlysis for the designted food types. Totl number of lbortories prticipting in nlysis of this food type.

FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 265 Tble 3. Interlbortory results for erobic plte counts for ground blck pepper smples (log10 CFU/g) by SimPlte 35, SimPlte 30, 35, nd 30 methods Lot A Lot B Lot C Test portion 1 Test portion 2 Test portion 3 Test portion 4 Test portion 5 Test portion 6 Lb 1 6.20 6.24 5.91 6.03 6.06 6.14 5.95 5.99 7.65 7.62 7.36 7.36 7.76 7.77 8.69 7.42 7.03 6.89 6.80 6.36 7.01 6.77 6.68 6.39 2 6.04 5.90 6.45 5.90 5.87 5.92 6.74 5.89 7.65 7.37 7.21 7.76 7.38 7.19 6.44 6.54 6.49 6.36 6.89 6.57 6.52 6.30 3 6.02 6.17 5.88 6.03 6.06 6.06 5.79 5.93 7.41 7.59 6.96 6.94 7.54 7.54 6.96 6.97 6.56 6.68 6.15 5.94 6.77 6.52 6.18 5.95 4 6.25 6.01 5.49 5.80 5.99 6.03 5.58 5.86 7.44 7.53 7.12 7.14 7.56 7.65 7.27 7.26 6.61 6.65 6.34 6.41 6.61 6.63 6.11 6.38 5 6.16 5.83 5.58 5.32 5.99 5.73 5.77 5.25 7.54 6.95 7.18 7.00 7.72 7.11 7.20 7.15 6.68 6.13 6.31 6.12 6.48 6.13 6.31 6.15 7 6.11 b 4.67 b 5.46 4.64 5.88 b 4.65 b 5.46 4.74 7.59 6.75 7.13 6.72 7.53 6.70 7.05 6.70 6.58 5.81 6.37 5.75 6.73 5.71 6.11 5.75 8 6.04 5.91 5.95 5.91 6.00 5.99 5.78 5.52 7.49 7.30 7.62 7.29 7.55 7.39 7.53 7.37 6.67 6.35 6.47 6.34 6.53 6.37 6.45 6.69 9 6.17 6.18 6.00 6.17 6.00 6.08 5.87 6.09 7.22 7.11 7.49 7.56 7.61 7.55 8.39 7.60 6.70 6.69 6.71 6.79 7.00 6.62 6.57 6.72 10 6.02 5.76 5.45 5.81 6.22 6.01 5.54 5.71 7.53 7.27 7.88 7.92 7.49 7.26 7.29 7.13 6.54 6.61 6.02 5.99 6.59 6.54 6.07 5.91 11 6.17 6.12 5.69 5.99 6.23 6.12 5.72 6.04 7.58 7.66 7.31 7.53 7.58 7.65 7.20 7.38 6.73 6.75 7.32 7.35 6.76 6.60 6.36 6.85 12 6.31 5.83 5.67 5.82 6.20 5.62 5.66 5.61 7.22 6.72 7.38 7.37 7.52 6.71 7.32 7.26 6.92 6.08 6.53 6.10 6.61 6.54 6.60 6.43 13 5.43 b 5.17 b 5.48 5.47 4.57 4.94 5.03 7.76 6.92 6.51 7.00 7.41 7.17 7.16 6.71 6.18 5.80 6.01 6.00 6.45 5.94 6.31 6.20 14 6.66 6.61 4.85 5.37 6.09 6.42 5.00 5.31 7.26 7.18 6.23 6.51 7.16 7.25 6.32 6.69 6.26 6.33 5.64 5.79 6.44 6.40 5.63 5.91 15 6.23 6.20 6.11 6.03 6.20 6.18 5.97 5.96 7.79 7.72 7.43 7.60 7.69 7.72 7.40 7.56 6.73 6.78 6.50 6.67 6.99 6.90 6.61 6.67 Counts were not in rnge. The pired test portion ws excluded from sttisticl nlysis. Outlier; dt not used in nlysis for method comprison of () nd () methods. b

266 FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 Tble 4. Interlbortory results for erobic plte counts for flour smples (log10 CFU/g) by SimPlte 35, SimPlte 30, 35, nd 30 methods Rye Whet White Test portion 1 Test portion 2 Test portion 3 Test portion 4 Test portion 5 Test portion 6 Lb 1 5.98 5.81 5.61 5.54 5.64 5.67 5.62 5.54 3.54 3.58 3.73 3.43 3.49 3.59 3.41 3.81 3.80 4.09 4.15 3.52 3.54 4.20 4.32 2 b b b b b b b b b b b b b b b b b b b b b b b b 3 5.66 5.63 5.97 5.99 5.38 5.40 6.00 5.97 3.20 4.32 4.15 3.52 4.28 4.20 4.59 4.26 4.69 4.68 4.20 4.04 4.72 4.75 4 5.57 5.66 5.56 5.52 5.52 5.63 5.40 5.52 3.74 3.57 3.40 3.61 3.90 3.68 3.08 3.51 4.04 4.15 4.36 4.28 4.26 4.08 4.00 4.15 5 6.04 5.23 5.98 5.26 5.98 5.20 5.82 5.28 4.43 4.08 3.89 3.47 4.29 3.86 3.76 3.30 4.80 4.00 4.51 3.92 4.86 4.63 4.48 3.93 6 5.88 5.79 c 5.88 5.87 c 6.00 5.65 c 6.84 6.86 c 4.26 3.83 3.96 3.88 4.26 3.81 3.28 3.53 4.43 4.20 4.54 4.40 4.46 4.36 4.58 4.34 7 5.67 5.93 5.72 5.76 5.69 5.82 5.98 6.00 3.20 3.60 4.11 3.99 3.62 3.93 3.89 3.90 3.91 4.00 c 4.52 4.84 c 3.66 4.32 c 4.34 4.34 c 8 5.79 5.86 5.81 5.86 5.94 5.88 6.26 5.83 3.23 3.64 3.58 3.85 3.08 3.57 3.85 3.71 4.36 4.23 4.43 4.26 3.23 3.11 4.26 4.26 9 5.86 6.08 6.08 5.94 6.08 6.08 6.11 6.08 4.08 4.11 4.00 4.00 4.08 4.08 3.97 4.04 4.23 4.20 4.57 4.38 4.32 4.23 4.63 4.56 10 5.73 5.58 5.47 4.69 5.99 4.95 5.74 5.20 3.71 3.46 4.56 4.09 4.11 4.79 4.03 4.56 4.53 4.11 3.52 3.53 4.23 4.08 11 5.89 5.86 6.00 6.04 5.86 5.86 6.00 6.04 4.45 4.48 4.23 4.11 4.56 4.61 4.32 4.20 4.81 4.53 4.86 4.80 4.82 4.68 4.97 4.78 12 6.01 5.90 5.32 5.18 5.81 5.57 5.60 5.36 3.81 4.06 3.18 4.04 3.76 3.45 3.41 4.67 4.34 4.09 4.34 4.02 4.06 13 6.23 5.89 5.83 5.93 6.61 6.04 5.76 5.86 4.43 3.56 3.72 4.45 3.61 3.61 3.76 4.62 3.83 4.30 4.04 4.58 4.11 4.40 4.08 14 5.15 5.82 5.83 5.96 5.72 5.77 5.83 6.04 2.90 3.90 3.97 3.00 3.81 3.93 4.11 4.04 4.65 4.48 4.08 4.08 4.74 4.64 15 5.78 5.82 5.59 5.79 5.91 5.98 6.00 5.88 4.10 3.81 3.49 3.63 3.52 3.81 3.94 3.91 4.61 4.45 4.51 4.40 4.41 4.41 4.45 4.26 Counts were not in rnge. The pired test portion ws excluded from sttisticl nlysis. Did not follow study instructions. Outlier; dt not used in nlysis for method comprison of () nd () methods. b c

FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 267 Tble 5. Interlbortory results for erobic plte counts for nut mets smples (log10 CFU/g) by SimPlte 35, SimPlte 30, 35, nd 30 methods Penuts Almonds Hzelnuts Test portion 1 Test portion 2 Test portion 3 Test portion 4 Test portion 5 Test portion 6 Lb 1 4.86 4.11 4.75 4.51 4.57 4.32 4.26 4.23 2.62 2.51 2.72 2.65 2.89 2.87 2.46 2.34 3.12 3.11 4.71 4.83 3.52 3.36 2.93 2.88 2 3.38 3.54 4.73 4.64 4.38 4.51 4.49 4.26 2.40 2.15 2.45 2.40 2.18 2.18 2.62 2.32 4.26 4.18 4.85 4.64 4.11 3.73 3.94 4.00 3 4.36 3.89 3.51 3.34 4.11 3.92 3.94 3.78 2.41 2.54 1.85 1.30 2.41 2.76 1.78 1.70 2.98 3.18 4.30 4.08 3.00 3.00 2.45 2.28 4 4.00 3.57 3.08 3.93 3.95 3.82 3.34 3.48 3.20 2.38 2.60 2.40 2.41 2.41 2.74 2.26 5.36 5.23 1.90 2.81 3.54 3.43 2.72 2.20 5 4.20 3.77 3.08 4.54 4.04 3.30 2.41 2.08 2.41 1.30 2.54 2.11 2.54 1.00 4.15 3.30 3.65 3.48 4.71 4.63 1.60 6 3.87 3.69 4.43 4.43 4.43 4.26 4.28 4.43 2.34 2.36 2.66 2.54 2.71 2.40 7 4.32 4.30 3.20 3.08 3.85 4.15 3.83 3.54 2.26 1.70 1.30 1.46 2.46 1.95 2.08 3.18 3.08 3.41 3.54 4.67 4.43 3.34 3.18 8 4.49 4.43 3.72 4.84 4.34 4.04 4.23 4.04 2.56 2.63 2.85 2.20 2.85 2.63 2.79 2.15 3.36 3.20 2.32 2.15 2.69 3.76 2.38 2.38 9 4.41 4.34 4.82 4.59 4.23 3.85 4.70 4.40 2.46 2.49 2.81 2.88 2.67 2.49 3.00 2.65 2.36 3.26 3.45 3.08 2.91 5.15 4.83 10 4.32 4.20 4.40 4.52 4.48 4.00 4.88 4.58 2.15 1.93 2.15 2.64 1.78 2.11 2.72 2.71 4.00 3.78 4.26 4.08 3.48 3.52 2.76 2.76 11 3.72 3.54 4.92 4.18 4.65 3.40 5.34 5.20 2.65 2.58 2.81 2.40 2.28 2.56 2.15 2.51 3.40 3.11 2.93 2.92 3.76 4.57 2.89 2.66 13 b b b b b b b b b b b b b b b b b b b b b b b b 14 3.40 4.15 4.94 5.18 4.68 4.74 5.08 6.04 2.76 2.80 2.81 2.83 2.84 2.84 2.85 2.86 3.20 3.20 4.26 4.34 3.18 3.26 3.26 3.30 15 4.32 4.20 4.66 4.54 4.32 4.23 3.11 3.67 2.63 2.60 2.54 2.64 2.86 2.72 3.02 2.99 4.11 3.79 5.46 4.59 4.68 3.11 2.87 Counts were not in rnge. The pired test portion ws excluded from sttisticl nlysis. Did not follow study instructions. b

268 FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 Tble 6. Interlbortory results for erobic plte counts for frozen hmburger ptties smples (log10 CFU/g) by SimPlte 35, SimPlte 30, 35, nd 30 methods Lot A Lot B Lot C Test portion 1 Test portion 2 Test portion 3 Test portion 4 Test portion 5 Test portion 6 Lb 1 2.79 2.74 2.91 3.12 2.79 2.76 2.90 3.10 3.94 3.81 3.93 3.95 3.81 3.59 3.88 4.90 2.41 2.64 2.68 3.15 2.54 2.62 2.72 3.08 2 2.51 2.58 2.83 2.63 2.46 b 2.92 2.83 3.70 3.11 5.07 4.72 3.43 3.27 4.99 4.85 2.36 b 2.99 2.94 2.38 2.54 2.90 2.95 3 3.48 2.95 3.28 3.36 3.11 2.73 3.14 3.35 4.24 4.11 5.25 5.01 4.23 3.75 4.86 5.03 2.85 2.69 2.94 3.18 3.13 2.70 2.54 3.09 4 3.47 3.05 3.26 3.10 3.16 3.11 3.32 3.42 4.00 4.02 5.37 5.30 3.86 3.81 5.35 5.39 3.39 3.25 3.20 3.13 3.89 3.45 3.32 3.15 5 2.61 2.53 2.96 3.04 3.39 2.66 3.09 2.91 4.58 4.05 5.37 4.95 4.00 2.91 5.30 4.96 2.54 2.57 b b 2.68 2.59 3.18 3.00 6 3.72 2.92 3.49 3.32 3.16 3.01 3.26 3.13 4.83 4.63 b b 4.81 4.42 b b 3.01 2.89 b b 2.75 2.62 4.87 5.86 7 2.81 2.77 3.02 2.81 2.32 b 2.90 2.90 2.96 3.04 4.99 4.88 2.99 2.79 4.95 4.91 2.41 2.54 2.84 2.86 2.59 2.58 2.49 2.78 8 2.76 2.85 3.48 3.28 3.18 2.82 2.94 3.13 4.25 4.42 3.86 5.14 4.31 4.44 3.94 5.25 2.38 2.81 3.08 3.16 2.71 2.90 3.04 3.05 9 3.39 3.41 3.19 3.40 3.48 3.46 3.40 3.81 4.31 4.16 5.37 5.56 4.12 3.91 5.50 5.54 3.08 3.06 3.34 3.63 2.36 2.57 3.30 3.54 10 3.48 2.69 c 3.22 3.90 c 3.61 2.60 c 3.52 2.77 c 4.37 3.19 5.54 5.10 4.32 4.03 5.48 5.26 2.95 2.99 3.01 2.79 2.95 3.09 3.21 2.96 11 3.03 3.23 3.19 3.23 3.12 3.26 3.06 3.13 4.21 3.99 5.44 5.20 4.15 3.94 5.30 5.16 3.02 3.38 3.32 2.95 2.99 3.11 2.94 3.08 12 3.44 3.01 3.06 3.06 3.19 b 3.14 3.12 4.11 4.18 5.01 5.09 4.66 4.86 5.19 5.21 2.80 2.72 2.74 3.11 2.97 3.04 2.70 3.02 13 3.43 2.87 3.46 3.24 3.46 2.73 3.38 3.16 4.69 4.33 5.25 5.43 4.53 4.12 5.25 5.38 3.16 2.82 2.98 3.13 3.18 2.91 2.93 3.13 14 2.43 2.69 2.63 3.14 2.45 2.63 2.81 3.26 3.22 3.29 4.61 5.40 3.33 3.30 4.66 5.44 2.73 2.88 3.05 3.13 2.71 2.85 3.04 3.18 15 2.89 2.92 3.18 3.22 3.19 3.44 3.34 3.39 4.01 3.72 5.45 5.37 3.89 3.89 5.44 5.44 3.03 2.92 3.17 3.11 3.24 3.03 3.21 3.18 Outlier; dt not used in nlysis for method comprison of () nd (). Counts were not in rnge. The pired test portion ws excluded from sttisticl nlysis. Outlier; dt not used in nlysis for method comprison of () nd (). b c

FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 269 Tble 7. Interlbortory results for erobic plte counts for frozen fruits smples (log10 CFU/g) by SimPlte 35, SimPlte 30, 35, nd 30 methods Peches Strwberries Blueberries Test portion 1 Test portion 2 Test portion 3 Test portion 4 Test portion 5 Test portion 6 Lb 1 2.83 3.10 3.45 2.99 3.04 3.29 3.04 3.15 3.26 3.28 3.41 2.80 2.92 2.81 2.79 3.04 2.91 2.67 2.63 2 2.98 2.97 3.74 3.74 2.99 3.07 3.50 3.68 4.26 3.72 4.28 b 4.40 3.97 4.18 b 2.26 2.61 2.58 2.54 3 3.53 2.87 4.25 3.67 3.47 3.00 3.93 3.97 4.40 3.94 4.22 4.00 4.46 4.13 4.09 3.99 3.51 3.11 2.49 2.72 3.18 2.87 2.36 2.53 4 4.01 3.02 3.60 3.19 3.35 4.06 3.41 4.00 3.50 3.89 4.84 3.86 3.44 3.90 4.81 3.05 2.66 2.74 2.48 2.94 2.81 2.97 2.94 5 2.86 3.67 3.23 2.92 3.63 3.28 4.26 3.75 3.97 3.79 4.19 3.44 4.19 3.87 2.61 2.61 2.24 2.61 2.54 2.95 2.63 6 3.23 3.20 4.13 4.11 2.93 3.06 4.30 4.13 4.86 4.16 4.05 3.78 4.65 4.20 4.13 3.79 3.20 2.66 3.06 2.64 3.35 2.93 2.73 2.62 7 2.18 c 2.99 c 3.50 3.39 3.61 c 3.44 c 3.54 3.58 4.13 4.31 4.06 3.88 2.68 2.28 3.17 2.48 2.70 8 d d d d d d d d d d d d d d d d d d d d d d d d 9 3.35 3.50 4.03 4.04 3.54 3.58 4.16 4.05 4.18 3.90 4.15 3.96 4.19 4.11 4.27 3.83 3.22 2.69 2.41 2.67 3.39 3.15 3.03 2.95 10 4.39 4.11 2.62 3.56 4.63 2.79 4.20 3.99 4.16 3.93 4.48 4.15 4.17 3.92 2.83 2.85 2.28 2.15 2.41 11 3.30 3.06 3.76 3.98 3.47 3.43 4.20 3.95 4.09 4.02 4.22 3.91 4.26 4.07 4.49 4.23 3.24 3.03 3.14 2.80 3.23 3.05 3.41 2.85 12 3.60 3.62 4.06 4.24 3.65 3.76 4.13 4.11 4.46 4.01 3.82 3.44 4.18 3.60 4.18 3.46 2.53 2.23 2.39 2.65 2.51 2.40 2.17 13 3.87 3.36 3.65 4.05 3.46 3.29 3.91 4.02 4.36 3.87 4.18 3.63 4.48 3.96 4.02 3.60 2.63 2.88 2.53 2.83 2.69 2.42 14 2.36 3.61 3.63 2.32 3.98 3.84 4.53 4.42 4.36 4.17 4.36 4.23 4.02 3.98 2.76 2.75 2.67 2.73 2.77 2.68 2.81 2.81 15 3.36 3.18 4.26 4.14 3.51 3.54 4.06 4.08 4.37 4.22 4.15 3.94 4.40 4.18 4.29 4.06 3.46 3.16 3.15 2.94 3.10 2.88 3.26 2.90 Counts were not in rnge. The pired test portion ws excluded from sttisticl nlysis. Spreding microorgnisms; unble to enumerte pltes. c Outlier; dt not used in nlysis for method comprison of () nd (). Did not follow study instructions. b d

270 FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 Tble 8. Interlbortory results for erobic plte counts for fresh vegetbles smples (log10 CFU/g) by SimPlte 35, SimPlte 30, 35, nd 30 methods Crrot Broccoli Celery Test portion 1 Test portion 2 Test portion 3 Test portion 4 Test portion 5 Test portion 6 Lb 1 6.20 6.18 7.41 7.64 7.18 7.19 7.24 7.64 6.68 6.62 6.68 7.18 6.91 6.84 7.18 8.00 7.04 7.06 6.90 8.47 7.54 7.33 7.67 8.66 2 7.42 7.79 7.74 7.62 7.56 7.74 7.81 7.62 7.13 7.35 7.27 7.26 7.49 7.64 7.92 8.18 8.67 8.81 8.65 8.81 3 7.42 7.45 7.59 7.59 7.43 7.53 7.69 7.59 7.01 7.01 7.74 7.69 7.43 7.41 7.92 7.73 7.70 8.38 8.63 8.72 8.67 8.66 8.58 4 8.45 8.80 8.82 8.17 8.31 5 7.08 6.39 6.78 6.59 7.22 6.19 6.94 6.59 7.03 5.87 7.90 7.39 6.96 5.70 7.81 7.22 7.34 6.30 7.70 7.88 6.97 6.03 8.36 8.04 6 7.50 7.21 8.10 7.65 7.70 7.43 7.88 7.65 7.44 7.23 8.27 8.15 7.38 7.01 8.04 7.94 7.67 7.32 8.85 8.87 7.65 7.24 8.77 8.83 7 7.03 7.03 7.10 7.05 6.69 6.83 7.40 7.05 6.57 6.60 7.76 7.67 6.37 7.48 7.79 7.70 6.89 6.77 8.17 8.20 6.52 6.67 8.22 8.25 8 7.30 7.90 8.15 7.92 7.40 7.90 8.45 7.92 7.29 7.76 8.09 7.38 6.94 7.49 8.03 7.48 7.25 8.12 7.98 7.27 7.87 9.81 9.39 15 7.40 7.41 8.07 7.83 7.65 7.45 7.89 7.83 7.07 7.01 8.53 8.35 7.11 7.07 8.35 7.97 7.73 7.51 9.07 8.98 7.78 7.65 8.97 8.95 16 7.23 7.18 8.00 7.88 7.42 7.37 7.81 7.88 7.15 7.09 7.91 8.03 7.52 7.40 8.40 8.32 7.65 7.54 9.19 9.00 7.73 7.56 8.76 8.90 17 7.58 7.73 7.76 7.93 7.82 8.05 7.89 7.93 7.38 7.77 8.44 8.29 7.45 7.69 8.58 8.39 7.68 8.54 9.20 9.00 7.49 8.42 8.81 8.87 18 7.56 7.41 5.53 5.82 8.28 7.96 7.20 5.82 7.73 7.38 7.57 7.78 7.91 7.76 7.97 7.98 7.32 8.16 8.42 7.81 8.50 19 6.23 6.35 5.60 6.88 6.68 5.60 4.30 5.90 4.30 6.40 6.32 4.60 6.90 7.42 4.30 8.41 7.56 Counts were not in rnge. The pired test portion ws excluded from sttisticl nlysis.

FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 271 the 3 lots nlyzed. The method reported better SD r vlues for Lot B test portions, but were not significntly different for Lots A nd C. The SD R vlues were not significntly different for the 3 lots nlyzed (Tble 2002.07C). Flour Three types of flour (rye, whet, nd white) were nlyzed (Tble 4). Lbortory 2 did not follow study instructions. The dt from this lbortory were excluded from sttisticl nlysis. Throughout the nlysis of flour, some lbortories reported erobic plte counts for certin test portions tht were not in the suitble counting rnge for or methods (Tble 4). Dt from these pired test portions were not used for sttisticl nlysis. Dt generted by the 35 nd the SimPlte 35 methods were compred sttisticlly. Overll, there were 11, 13, nd 14 lbortories tht submitted vlid dt for whet, white, nd rye flour test portions, respectively. The men log counts from the 35 nd the SimPlte 35 methods were not significntly different for the 3 lots nlyzed. The method reported better SD r nd SD R vlues for whet test portions (p < 0.05). The SD r nd SD R vlues for rye nd white test portions were not significntly different between the 2 methods (Tble 2002.07A). Dt generted by the 30 nd SimPlte 30 methods were compred sttisticlly. Overll, 14 lbortories submitted vlid dt for ll 3 types of flour for sttisticl nlysis. The men log counts from the nd the SimPlte methods were not significntly different for the 3 lots nlyzed. The method reported better SD r vlues for the whet test portions. However, the SD R ws not significntly different between the 2 methods for whet test portions. The SD R vlues for rye test portions were significntly better for the SimPlte method (p > 0.05). The white flour test portions reported similr SD r nd SD R vlues between the 2 methods (Tble 2002.07B). Dt generted by the 30 nd the 35 methods were compred sttisticlly. Lbortories 6 nd 7 were determined to be outliers by the Cochrn test for rye flour nd white flour test portions, respectively. These pired dt were excluded from sttisticl nlysis. Overll, there were 10, 12, nd 13 lbortories tht submitted vlid dt for whet, white, nd rye flour test portions, respectively. Men log counts between the 2 methods were similr for rye nd whet flour test portions. The method recovered higher men log counts for white flour test portions (p < 0.05). The SD r nd SD R vlues of the 2 methods were not significntly different for the rye nd whet test portions nlyzed. The method reported better SD r vlues for white flour test portions. However, the SD R vlues were not significntly different for white flour test portions (Tble 2002.07C). Nut Mets Three types of nut mets (penuts, lmonds, nd hzelnuts) were nlyzed (Tble 5). Lbortory 13 did not follow study instructions. The dt from this lbortory were excluded from sttisticl nlysis. Collbortors reported higher incidence of low erobic plte counts tht were not in the suitble counting rnge for 35 or 30 methods for lmond test portions. There my hve been die off of microorgnisms during shipment before nlysis. All dt reported for lmond test portions were used for sttisticl nlysis. For penuts nd hzelnuts, some lbortories reported erobic plte counts for certin test portions tht were not in the suitble counting rnge for or methods (Tble 5). Dt from these pired test portions were not used for sttisticl nlysis. Dt generted by the 35 nd the SimPlte 35 methods were compred sttisticlly. Overll, 13 lbortories submitted vlid dt for penut nd lmond test portions, nd 12 lbortories submitted vlid dt for hzelnut test portions. The men log counts for the nd the SimPlte methods were not significntly different for the lmond nd hzelnut test portions nlyzed. Higher men log counts were recovered by the SimPlte method (p < 0.05) for penut test portions. The penut nd lmond test portions reported better SD r vlues for the method, wheres the hzelnut test portions reported similr SD r vlues between the 2 methods. The SD R vlues of the 2 methods were not significntly different for ny of the 3 nut mets nlyzed (Tble 2002.07A). Dt generted by the 30 nd SimPlte 30 methods were compred sttisticlly. Overll, 12 lbortories submitted vlid dt for penuts nd lmonds, nd 11 lbortories submitted vlid dt for hzelnuts for sttisticl nlysis. Men log counts between the 2 methods were equivlent for ll 3 lots of nut met test portions. The method reported better SD r vlues for lmonds test portions (p < 0.05). The SD r vlues were not significntly different between the 2 methods for penut nd hzelnut test portions. The SD R were not significntly different between the 2 methods for ll 3 lots nlyzed (Tble 2002.07B). Dt generted by the 30 method nd the 35 method were compred sttisticlly. Overll, 12 lbortories submitted vlid dt for ll 3 types of nut mets nlyzed. Men log counts between the 2 methods were equivlent for ll 3 lots of nut met test portions. The method reported better SD r vlues for lmond test portions. The SD R vlues were significntly better for the method for penut nd lmond test portions (p < 0.01). However, the SD r nd SD R vlues were not significntly different between the 2 methods for the hzelnut test portions (Tble 2002.07C). Frozen Hmburger Ptties Three lots of frozen hmburger ptties (Lots A C) were nlyzed (Tble 6). Throughout the nlysis of frozen hmburger ptties, some lbortories reported erobic plte counts for certin test portions tht were not in the suitble counting rnge for or methods (Tble 6). Dt from these pired test portions were not used for sttisticl nlysis. Dt generted by the 35 nd the SimPlte 35 methods were compred sttisticlly. Overll, 15 lbortories submitted vlid dt for ll 3 lots of ptties nlyzed. The men log counts between the nd the SimPlte methods were not significntly different for the 3 lots nlyzed. The method reported better repetbility for Lots A

272 FELDSINE ET AL.: JOURNAL OF INTERNATIONAL VOL. 86, NO. 2, 2003 nd C, wheres the SimPlte method reported better repetbility for Lot B. Lots A nd C generted better SD R vlues for the method, wheres for Lot B, the SD R ws not significntly different between the 2 methods (Tble 2002.07A). Dt generted by the 30 nd SimPlte 30 methods were compred sttisticlly. Lbortory 10 ws determined to be n outlier for Lot A by the Cochrn test; therefore, the pired dt were excluded from sttisticl nlysis. Overll, there were 14, 13, nd 15 lbortories tht submitted vlid dt for Lots A C test portions, respectively. The men log counts from the nd the SimPlte methods were not significntly different for the 3 lots nlyzed. The method reported better SD r for Lots B nd C test portions. The SD r ws not significntly different for Lot A between the 2 methods. The method reported better SD R for Lot B test portions. The SD R ws not significntly different for Lots A nd C between the 2 methods (Tble 2002.07B). Dt generted by the 30 nd the 35 methods were compred sttisticlly. Lbortory 10 ws determined to be n outlier by the Cochrn test for Lot A; therefore, the pired dt were excluded from sttisticl nlysis. Overll, 14 lbortories submitted vlid dt for Lots A nd B test portions, nd 15 lbortories submitted vlid dt for Lot C test portions. The method recovered higher men log counts for ll 3 lots of ptties nlyzed (p < 0.01). The method reported better SD r for Lots B nd C test portions. The SD r ws not significntly different for Lot A between the 2 methods. The method reported better SD R for Lot B test portions. The methods were not significntly different for Lot A, nd the method reported better SD R for Lot C (Tble 2002.07C). Frozen Fruits Three types of frozen fruits (peches, strwberries, nd blueberries) were nlyzed (Tble 7). Lbortory 8 did not follow study instructions. The dt from this lbortory were excluded from sttisticl nlysis. Throughout the nlysis of frozen fruits, severl lbortories reported erobic plte counts for certin test portions tht were not in the suitble counting rnge for 35 or 30 methods (Tble 7). Dt from these pired test portions were not used for sttisticl nlysis. Dt generted by the 35 nd the SimPlte 35 methods were compred sttisticlly. Lbortory 7 ws determined to be n outlier by the Cochrn test for pech test portions; therefore, the pired dt were excluded from sttisticl nlysis. Overll, 10 lbortories submitted vlid dt for peches nd blueberries, nd 12 lbortories submitted vlid dt for strwberries. The SimPlte method recovered higher men log counts for strwberries nd blueberries (p < 0.01). The SD r nd SD R vlues of the 2 methods were not significntly different for ny of the 3 fruits nlyzed (Tble 2002.07A). Dt generted by the 30 nd SimPlte 30 methods were compred sttisticlly. Overll, 13 lbortories submitted vlid dt for peches nd blueberries, nd 11 lbortories submitted vlid strwberry dt for sttisticl nlysis. Strwberries recovered higher men log counts by the SimPlte method (p < 0.05). The method reported better SD r vlues for pech nd strwberries test portions. However, the SimPlte method showed better SD R vlues for these 2 fruits. The SD r nd SD R vlues for blueberries were not significntly different between the 2 methods (Tble 2002.07B). Dt generted by the 30 nd the 35 methods were compred sttisticlly. Lbortory 2 ws unble to record counts from the 30 pour pltes becuse of spreding microbil growth. These pired test portions were excluded from sttisticl nlysis. Overll, 11 lbortories submitted vlid dt for peches nd strwberries, nd 10 lbortories submitted vlid dt for blueberries. Men log counts from the pech test portions were higher for the method (p < 0.01), wheres the method recovered higher men counts from the blueberry test portions (p < 0.05). The method reported better SD r vlues for pech nd strwberry test portions. The SD R vlues were not significntly different for the 3 lots of frozen fruits nlyzed (Tble 2002.07C). Fresh Vegetbles Three types of vegetbles (crrots, broccoli, nd celery) were nlyzed (Tble 8). Throughout the nlysis of fresh vegetbles, some lbortories reported erobic plte counts for certin test portions tht were not in the suitble counting rnge for 35 or 30 methods (Tble 8). Dt from these pired test portions were not used for sttisticl nlysis. Dt generted by the 35 nd the SimPlte 35 methods were compred sttisticlly. Overll, 12 lbortories submitted vlid dt for crrot nd broccoli test portions, nd 10 lbortories submitted vlid dt for celery test portions. The men log counts between the 35 nd the SimPlte 35 methods were not significntly different for the 3 types of vegetbles nlyzed. The SD r nd SD R vlues for crrot test portions were not significntly different between the 2 methods. The SimPlte method reported better SD r nd SD R vlues for broccoli test portions. The celery test portions reported better SD R vlues for the SimPlte method (p < 0.01; Tble 2002.07A). Dt generted by the 30 nd SimPlte 30 methods were compred sttisticlly. Overll, 11 lbortories submitted vlid dt for crrot nd celery test portions, nd 13 lbortories submitted vlid dt for broccoli test portions. The men log counts between the 30 nd the SimPlte 30 methods were not significntly different for the 3 types of vegetbles nlyzed. The SD r nd SD R vlues were not significntly different between the 2 methods for crrot nd broccoli test portions. The method reported better SD r for the celery test portions. The SD R, however, ws not significntly different between the 2 methods for the celery test portions (Tble 2002.07B). Dt generted by the 30 nd the 35 methods were compred sttisticlly. Overll, 11 lbortories submitted vlid dt for crrot nd broccoli test portions, nd 9 lbortories submitted vlid dt for celery test portions. The method recovered higher men log counts for the broccoli nd celery test portions (p < 0.01). Men log counts for crrot test