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townsend naqui journal of aoac international vol 81 no 3 1998 563 food biological contaminants comparison of simplate total plate count test with plate count agar method for detection and ...

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                                                                   TOWNSEND & NAQUI: JOURNAL OF AOAC INTERNATIONAL VOL. 81, No. 3, 1998 563 
                FOOD BIOLOGICAL CONTAMINANTS 
                Comparison of SimPlate Total Plate Count Test with Plate Count 
                Agar Method for Detection and Quantitation of Bacteria in Food 
                DAVID E. TOWNSEND and ALI NAQUI 
                IDEXX Laboratories, Inc., One IDEXX Dr, Westbrook, ME 04092-2041 
                                                                                                                                                              Downloaded from https://academic.oup.com/jaoac/article/81/3/563/5683999 by guest on 13 September 2022
                The SimPlate™ Total Plate Count (TPC) test, devel-                   canted, the SimPlates are placed in a dry incubator for 24 h. The 
                oped by IDEXX Laboratories, Inc., detects and                        medium detects foodborne bacteria by testing for the presence 
                quantitates total bacterial concentration in food af-                of key enzymes common in these bacteria. If the enzyme is 
                ter 24 h of incubation. The performance of Sim-                      present, a blue fluorescent color (4-methylumbelliferone) is 
                Plate TPC was compared with that of the plate                        produced in the incubating wells. This reaction should not be 
                count agar (PCA) method for enumerating total bac-                   confused with the MUG reaction used to specifically detect Es-
                terial concentration of 255 food samples repre-                      cherichia coli in food and environmental samples (4), although 
                senting 15 different food matrixes. Total bacterial                  the principle is the same. Multiple enzyme substrates present in 
                counts on SimPlate TPC were measured after 24 h                      the TPC medium target different bacterial enzymes. Not all 
                of incubation and plotted against values obtained                    bacteria have each of these enzyme activities but they should 
                from PCA after 48 h. Simple regression analysis of                   have at least one. The activity of only one targeted bacterial 
                the data showed strong correlation between the                       enzyme is sufficient for detection to occur. This multiple en-
                methods (r = 0.95); the sensitivity of SimPlate TPC                  zyme technology was developed at IDEXX Labs, Inc., West-
                for foodborne bacteria was 96% relative to PCA                       brook, ME, and is patent-pending (5). The number of blue fluo-
                (slope = 0.96). It was concluded that SimPlate TPC                   rescent wells present after 24 h of incubation is converted into 
                is a suitable alternative for the detection and quanti-              the MPN of bacteria present in that food sample. The blue fluo-
                tation of foodborne bacteria. The method has been                    rescent color is easy to see and permits a rapid, accurate, and 
                granted Performance Tested Certification by the                      consistent count. It is clearly distinguishable from food particu-
                AOAC Research Institute.                                             lates which often complicate the reading of existing colony 
                                                                                     count methods. Furthermore, swarming bacilli, which tend to 
                                                                                     populate processed food, do not interfere with the reading of 
                       imPlate Total Plate Count (TPC) allows quantitation of        this test as they do with other standard methods (2). 
                       total bacterial concentration in food after 24 h of incuba-      The procedure of setting up and reading a SimPlate for the 
                Stion. This represents a significant time savings advantage          TPC test is very different from traditional aerobic plate count 
                over other methods such as conventional plate count agar             methods. With SimPlate, foodborne bacteria are suspended in 
                (PCA), AOAC 966.23, which requires a 48 h incubation period          the TPC medium and separately compartmentalized into wells 
                 (1). Internal testing has demonstrated that SimPlate TPC can be     where biochemical activities and not growth into colonies de-
                used to detect and quantitate total bacterial concentration in all   termine if viable bacteria are present. Detection by this bio-
                foods except raw liver, raw cheese products, unprocessed flour,      chemical process is key for the relatively short incubation pe-
                raw nuts, and thyme which contain interfering substances (2).        riod, because it takes fewer bacteria to produce a detectable 
                TPC medium is purchased as a pre-made sterile powder and             signal in a SimPlate well than are required to form a clearly 
                 added to sterile diluent to obtain a source of bulk medium. Sim-    visible colony. Quantitation by SimPlate also differs from tra-
                Plates are autoaliquoting patented incubation vessels for the        ditional methods in that the number of positive wells and not 
                 TPC medium and support quantitation of foodborne bacteria by        colony forming units (CFU) determines the final bacterial 
                 most probable number (MPN) methodology (3). SimPlates               count. The relationship between the number of positive wells 
                 come in 2 sizes: normal, with a maximum counting range of           and the final MPN is not linear but is rooted in the well-estab-
                 738; and high, with a maximum counting range of 1659.               lished mathematical principle of the Poisson distribution (6). 
                    Prepared food samples are placed on the center loading pad       Poisson distribution, in this case, relates to distribution of bac-
                 of a SimPlate followed by an overlay of TPC liquid medium.          teria into the SimPlate wells. The total number of wells present 
                 The liquid is distributed into a fixed number of individual incu-   in each SimPlate determines their maximum counting range, 
                 bating wells (84 for normal counting range SimPlate and 198         and the number of positive wells determines the MPN. 
                 for high counting range SimPlate). After excess liquid is de-          Previous work has demonstrated that results obtained by 
                                                                                     SimPlate TPC after 24 h of incubation are highly correlated 
                    Received October 17, 1997. Accepted by AH January 9, 1998.       with the 48 h results obtained by the PCA, Petrifilm™ Aerobic 
                 564 TOWNSEND & NAQUI: JOURNAL OF AOAC INTERNATIONAL VOL. 81, No. 3, 1998 
                 Table 1. Food matrixes used in this study                              were fully randomized and labeled with a blind code so that the 
                 Category                             Specific food type                technician conducting the study did not know the type of food 
                                                                                        being tested. Solid food samples (25 g) were combined with 
                 Dry processed food blend               Nonfat dry milk                 225 mL sterile Butterfield's phosphate buffer (pH 7.2) and 
                 Dry spices                      Ethylene oxide-treated peper           pummeled in a stomacher for 2 min at normal setting. Liquid 
                 Produce                           Frozen mixed vegetables              foods were diluted directly in sterile Butterfieled's phosphate 
                 Raw meat                    Hamburger, chicken, and raw shrimp         buffer. Environmental swabs were suspended in 9.9 mL Butter-
                 Dairy                        Raw commingled milk, pasteurized          field's phosphate buffer and hand-massaged to dislodge bacte-
                                                 whole milk, dry infant formula,        ria from the swab into the buffer. Homogenates from 5 swabs 
                                                        and ice cream                   were combined for each lot tested. The procedure for diluting 
                 Confectionery                      Milk chocolate (no nuts)            homogenates and applying them to SimPlates and PCA dif-
                 Fruits                                   Apple juice                   fered depending on the anticipated population of bacteria in              Downloaded from https://academic.oup.com/jaoac/article/81/3/563/5683999 by guest on 13 September 2022
                 Pet food                              Dry and canned                                                    2     3         4
                                                                                        each sample. For raw milk, 10~ , 10 , and KT  dilutions were 
                 Environmental swabs                    Stainless steel                                                     _1
                                                                                        prepared; for all other liquids 10 , 10' , and 10" dilutions 
                                                                                        were prepared. 
                 Count plates, and Redigel™ Total Count methods for enumer-                SimPlate Procedure 
                 ating total bacterial concentration in food (2). These data,              TPC multiple test medium (2.6 g in 20 mL vials) supplied 
                 which were collected at 8 separate laboratories and represented        as part of the kit was hydrated in 100 mL sterile deionized water 
                 over 700 different food samples, consistently yielded correla-         and provided enough medium to perform 10 SimPlate TPC 
                 tion coefficients >0.96.                                               tests using the normal counting range SimPlate. Appropriate 
                    This report describes an independent study conducted under          dilutions of each food were prepared and 0.1-1 mL was placed 
                 the management of the AOAC Research Institute to verify the            on the center loading pad of the SimPlate. TPC medium was 
                 efficacy of the SimPlate TPC method for enumerating the total          then added to each SimPlate for a final volume (food sample + 
                 bacterial concentration of food.                                       medium) of 10 ± 0.2 mL. The mixture of sample and medium 
                 Experimental                                                           was distributed in all wells by gently swirling and tilting the 
                    Apparatus                                                           plate. Care was taken not to introduce air bubbles into the wells. 
                                                                                        Excess sample was decanted from the SimPlate by holding the 
                    (a) SimPlates.—Normal counting (738) and high counting              base of the SimPlate in one hand while lifting the cover with 
                 (1659) ranges (IDEXX Laboratories, Inc., Westbrook, ME).               the other hand and carefully pouring off excess liquid into a 
                    (b) Dry incubator.—-32° and 35°C.                                   collection container. The presence of food particles does not 
                    (c) Stomacher.—Tekmar, Cincinnati, OH.                              interfere with the inoculation, well filling, and decanting steps 
                    (d) Petri dishes.—100 mm diameter.                                  of the SimPlate procedure. 
                                                                                           Inverted SimPlates were incubated at 32°C for dairy and 
                    Culture Media and Reagents                                          35°C for nondairy foods for 24 h. The number of blue fluores-
                    (a) Dehydrated total plate count (TPC) medium. -IDEXX               cent wells in each SimPlate was recorded after excitation with 
                 Laboratories, Inc.                                                     a portable long wavelength (365 nm) UV light. The MPN/Sim-
                    (b) Plate count agar (PCA).—Difco Laboratories, Detroit, MI.        Plate was determined by using a SimPlate MPN table provided 
                    (c) Butterfield's phosphate buffer.—pH 7.2.                         by IDEXX Laboratories, Inc. The number of positive wells in 
                                                                                        each SimPlate device was counted, and the SimPlate MPN ta-
                    Experimental Protocol                                               ble was used to determine the MPN of the plate. The MPN table 
                                                                                        was constructed by following the same mathematical principles 
                    An experimental protocol for evaluating the SimPlate TPC            used by traditional 3- or 5-tube MPN methods (7). However, 
                 method for determining MPN of bacteria in commercial food              the SimPlate MPN method is much more accurate than tradi-
                 samples was developed by IDEXX Laboratories, Inc., along               tional MPN methods because of the large number, 84 or 198, 
                 with input from 2 expert reviewers working on behalf of the            of incubating wells in the SimPlate device. Thus, MPN deter-
                 AOAC Research Institute.                                               minations by SimPlate are highly correlated with colony count 
                    Sample Preparation                                                  methods (see below). A correction factor was included in each 
                                                                                        MPN table to account for the loss of sample during the decant-
                    Table 1 lists foods analyzed by the SimPlate TPC and PCA            ing step of the SimPlate procedure. For the normal counting 
                 methods. These foods were chosen because they represent                range SimPlate, the correction factor is 2 because of the loss of 
                 vastly different classes of raw and processed food products.           half of the sample during decanting. For the high counting 
                 Three different lots of each food matrix were tested by both           range SimPlate the correction factor is 1.8. In both tables the 
                 methods in 5 replicates, each representing one PCA and one             calculated MPN value was multiplied by the appropriate cor-
                 SimPlate test. Each replicate was made large enough to be ex-          rection factor to arrive at the final MPN value listed in each 
                 amined by both methods. One technician prepared the food               table. The MPN/g (mL) of food was calculated by dividing the 
                 samples and a second did the actual testing. All food samples          MPN/SimPlate by the inoculum volume, and then multiplying 
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...Townsend naqui journal of aoac international vol no food biological contaminants comparison simplate total plate count test with agar method for detection and quantitation bacteria in david e ali idexx laboratories inc one dr westbrook me downloaded from https academic oup com jaoac article by guest on september the tpc devel canted simplates are placed a dry incubator h oped detects medium foodborne testing presence quantitates bacterial concentration af key enzymes common these if enzyme is ter incubation performance sim present blue fluorescent color methylumbelliferone was compared that produced incubating wells this reaction should not be pca enumerating bac confused mug used to specifically detect es terial samples repre cherichia coli environmental although senting different matrixes principle same multiple substrates counts were measured after target all plotted against values obtained have each activities but they simple regression analysis at least activity only targeted data...

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