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1404 journal of food protection vol 62 no 12 1999 pages 1404 1410 copyright q international association of milk food and environmental sanitarians anewmediumfor determining the total plate count in ...

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                 1404
                 Journal of Food Protection, Vol. 62, No. 12, 1999, Pages 1404–1410
                 Copyright Q, International Association of Milk, Food and Environmental Sanitarians
                     ANewMediumfor Determining the Total Plate Count in Food
                                                                   C. F. SMITH AND D. E. TOWNSEND*
                                                 IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092–2041, USA
                                                             MS99-126: Received 27 April 1999/Accepted 6 July 1999
                                                                                 ABSTRACT                                                                                  Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/12/1404/1663823/0362-028x-62_12_1404.pdf by Indonesia user on 13 September 2022
                           SimPlate for Total Plate Count–Color Indicator (TPC-CI, IDEXX Laboratories, Inc., Westbrook, Me.) is a new medium
                      that incorporates the redox dye resazurin to detect and quantify bacteria in food. Enumeration is achieved by the most probable
                      number method using a SimPlate device. Viable bacteria are detected in each well of the SimPlate device by the biochemical
                      reduction of resazurin, which is blue, to the pink resorufin or the clear dihydroresorufin indicators. Results after 24 h of
                      incubation for TPC-CI are highly correlated with standard plate count agar after 48 h of incubation. Correlation coefficients
                      from studies conducted at five laboratories ranged from 0.94 to 0.98 in side-by-side comparisons against standard plate count
                      agar. Four additional test sites, using alternative methods for determining the aerobic plate count in food, reported similar
                      results in comparison studies (r 5 0.91 to 0.97). The slopes from linear regression analysis at all sites ranged from 0.91 to
                      0.98, with y intercepts ranging from 0.11 to 0.84. Samples used for the validation of TPC-CI included raw food products (i.e.,
                      liver and grains), which may contain natural enzymes that interfere with enzyme-based detection methods. No interference
                      was seen from the foods tested. These results suggest that TPC-CI is a suitable alternative to existing plate count methods
                      and has reduced incubation time.
                      Recent outbreaks of food poisoning have heightened
                 awareness of food microbiology and, in particular, the de-
                 velopment of rapid bacteriological tests. The mesophilic
                 aerobic plate count or standard plate count (SPC) of a fresh
                 food product reflects conditions of raw product quality, pro-
                 cessing, handling, and storage (5). The importance of SPC
                 is further reflected in standard recommendations for a num-
                 ber of food products (4). Because of its importance, there
                 is a need for the bacterial numbers in food to be known in
                 a period that permits rapid response to a food processing
                 problem. Current standard methods require a 48-h incuba-
                 tion period before results are known. In this article, we
                 describe a new method that permits accurate quantification
                 of the total bacterial count in food after only 24 h of in-
                 cubation. This method uses a bacterial growth medium con-
                 taining the redox dye resazurin to detect foodborne bacteria.             FIGURE 1. SimPlate showing positive (pink or beige) and neg-
                 Quantification is achieved by using a SimPlate device (2,                  ative (dark blue) wells.
                 3, 8, 9).
                      An earlier formulation for total plate count determi-
                 nations using the SimPlate device was based on multiple                   Count–Color Indicator (TPC-CI) does not use these enzyme
                 enzyme substrates that yielded a fluorescent signal when                   reactions to detect the presence of foodborne bacteria, and
                 metabolized by bacteria (2). This earlier method (SimPlate                therefore, this medium can be used to measure the total
                 for Total Plate Count) correlates well with standard meth-                bacterial count in foods without interference from the nat-
                 ods and has received ‘‘Performance Tested Certification’’                  urally occurring, endogenous enzymes present in the food
                 from the Association of Official Analytical Chemists Re-                   sample.
                 search Institute (9). The manufacturer of this medium                           TPI-CI medium comes as a presterilized powder and
                 (IDEXX Laboratories, Inc., Westbrook, Me.) specifically                    is reconstituted with sterile diluent before use. The sample
                 recommends certain food types not be tested with this                     to be tested is added to the SimPlate device followed by
                 method. This is because these foods (i.e., raw liver, wheat               reconstituted medium. The SimPlate device is incubated in
                 flour, and nuts) are known to contain enzymes that interfere               the same manner as a petri dish, but results are read after
                 with the performance of this test. SimPlate for Total Plate               24 h of incubation. Microbial growth is detected in indi-
                 * Author for correspondence. Tel: 207-856-0823; Fax: 207-856-0474;        vidual wells of the SimPlate device by a change in color.
                   E-mail: David-Townsend@idexx.com.                                       Growing bacteria reduce the dark blue resazurin to a pink
              J. Food Prot., Vol. 62, No. 12                   SIMPLATE FOR TOTAL PLATE COUNT–COLOR INDICATOR           1405
              TABLE 1. Foods tested internally at IDEXX laboratories
                     Food                             No. samples             Food                              No. samples
              Fresh produce                                            Dairy products
               Green pepper                                1             Ricotta cheese                              1
               Grapes                                      1             Swiss cheese                                1
               Salad greens                                1             American cheese                             1
               Alfalfa sprouts                             1             Cheese and bacon dip                        1
               Red cabbage                                 1             Cheddar cheese                              1
               Carrots                                     1             Monterey jack cheese                        1
               Broccoli                                    1             Cheddar cheese                              1
               Cauliflower                                  1           Frozen foods
               Apple                                       1             Beef stew                                   1             Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/12/1404/1663823/0362-028x-62_12_1404.pdf by Indonesia user on 13 September 2022
               Walnuts                                     1             Lasagna                                     1
               Cantaloupe                                  1             Corn                                        1
               Honey dew melon                             1             Chicken fettuccine                          1
               Fresh tomato                                1             Squash                                      1
               Potato, red-skinned                         1             Pasta and cheese dinner                     1
               Eggplant                                    1             Home fries, frozen                          1
               Mushrooms                                   1             Baked potato with broccoli                  1
               Onion                                       1             Pizza                                       1
               Sweet potato                                1             Breaded haddock                             1
               White potato, raw                           1             Bean and cheese burrito                     1
               Cabbage                                     1             Chicken pot pie                             1
               Alfalfa and radish sprouts                  2
               Broccoli sprouts                            1           Prepared foods and mixes
               Clover sprouts                              1             Potato salad                                1
               Almonds                                     1             Muffin mix with chocolate                    1
              Meats and seafood                                          Pancake mix                                 1
               Beef liver                                  2             Fudge brownie mix                           1
               Chicken liver                               1             Oat muffin mix                               1
               Ground beef                                 6             Rice                                        1
               Ground chuck                                1             Flavor packet for rice                      1
               Ground turkey                               2             Milk chocolate bar                          1
               Sausage                                     3             Lemon ice tea cooler                        1
               Ground chicken                              1             Cake mix                                    1
               Chicken breast                              2             Orange elixir                               1
               Whole chicken legs                          1             Chocolate raisins                           1
               Raw chicken strips                          1             Croutons                                    1
               Chicken thigh                               1             Salad dressing, ranch                       1
               Chicken drumsticks                          1             Instant potatoes                            1
               Ground pork                                 2             Mushroom gravy mix                          1
               Pork strips, raw                            1             Corn cereal                                 1
              Dairy products                                             Oat cereal                                  1
               Raw milk                                   28             Ready-to-serve chicken strips               1
               Pasteurized milk                            1             Peanut butter                               1
               Mint chip ice cream                         1             Butterscotch pudding mix                    1
               Coffee fudge frozen yogurt                  1             Flavored gelatin                            1
               Butter pecan ice cream                      1             Chicken soup mix                            1
               Raspberry sherbet                           1             Ramen soup mix                              1
               Lime sherbet                                1             Strawberry-mango nectar                     1
               Orange sherbet                              1             Stuffing mix                                 1
               Vanilla ice cream                           1             Flavor packet for stuffing                   1
               Chocolate ice cream                         1             Cocoa                                       1
               Strawberry ice cream                        1             Marshmallows, dehydrated                    1
               Dry milk                                    2           Dry goods
               Nondairy creamer                            2             Egg noodles                                 1
               Cream cheese                                1             Onion powder                                1
               Cream—half and half                         1             Cornmeal                                    2
               Whipping cream                              1             Blue corn meal                              1
               Egg                                         1             Cornstarch                                  1
               Egg substitute                              1             White flour                                  1
               Butter                                      1             Whole wheat flour                            1
               Margarine                                   1             Oatmeal                                     1
               Cheese spread slices                        1             Dog food                                    1
             1406         SMITH AND TOWNSEND                                                          J. Food Prot., Vol. 62, No. 12
             TABLE 2. Foods tested against standard plate count agar, Petrifilm, Redigel, or orange serum agar in field trials
                    Food                              No. samples             Food                             No. samples
             Fresh vegetables                                         Prepared foods
               Alfalfa sprouts                             1            Beef ravioli                                1
               Apples                                      3            Cannelloni mix                              1
               Bean sprouts                                1            Chicken cacciatore                          1
               Broccoli                                    6            Macaroni and cheese                         3
               Cabbage                                     4            Pasta salad                                 1
               Carrots                                     3            Pesto                                       4
               Cauliflower                                  3            Primavera mix                               1
               Diced celery                                1            Rigatoni arrabata                           1              Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/12/1404/1663823/0362-028x-62_12_1404.pdf by Indonesia user on 13 September 2022
               Diced cole slaw                             1            Spicy meat sauce                            1
               Diced peppers                               1            Stir-fry mix                                1
               Eggplant                                    3            Sun-dried tomato pesto                      2
               European salad mix                          1            Traditional lasagna                         1
               Frozen, fine-cut basil                       1            Veal tortellini mix                         1
               Iceberg lettuce                             1          Frozen foods
               Lettuce                                     5            Frozen pot pie                              6
               Minced garlic                               1            Frozen ravioli                              6
               Onions                                      4            Frozen garden burger                        6
               Parsley                                     1            Frozen fish sticks                           2
               Potatoes                                    3
               Red cabbage                                 1          Dry goods
               Romaine lettuce                             1            Cake mix                                    6
               Shredded carrots                            1            Egg noodles                                 6
               Sliced mushrooms                            1            Flour                                       6
               Spinach                                     1            Muffin mix                                   6
               Vegetable mix                               1            Oat cereal                                  2
               Whole tomatoes                              1            Pancake mix                                 6
             Juices                                                     Pasta                                       6
               Apple juice                                 5            Pretzels                                    1
               Cranberry apple                             2          Dairy
               Frozen apple juice                         11            Butter fat (60%)                            2
               Grapefruit juice                            8            Butter/oil blend                            5
               Juice cocktails                             2            Buttermilk powder                           2
               Lemon juice                                 3            Condensed skim milk                         1
               Lemonade                                    6            Cream                                       1
               Orange juice                                7            Cream, 35%                                  1
               Pasteurized blends                         31            Dry milk                                    6
               Raw juice blends                           18            Evaporated skim milk                        1
               Tropical punch                              2            Four-cheese blend                           1
             Meats and fish                                              Frozen sherbet                              6
               Catfish                                      2            Ice cream, chocolate                        6
               Chicken breast fillet                        1            Ice cream, vanilla                          6
               Chicken parts                               7            Liquid whole eggs                           1
               Chicken salad                               1            Raw milk                                    1
               Ground beef                                 9            Skim milk                                   8
               Ground pork                                 6            Skim milk powder                            7
               Ground turkey                               6            Whole milk                                  8
               Pork fritter                                1          Miscellaneous
               Pork loin                                   7            Black pepper                                1
               Pork sausage                               12            Chiller water                               1
               Sausage                                     7            Marsala wine sauce                          2
               Steak                                       7            Red pepper sauce                            1
               Turkey parts                                6            Salsa                                       1
             Prepared foods                                             Sausage gravy                               1
               Agnolotti and cheese                        1            Seafood sauce                               1
               Agnolotti and marinara                      2            Slivered almonds                            2
               Alfredo sauce                               1            Tartar sauce                                1
                 J. Food Prot., Vol. 62, No. 12                              SIMPLATE FOR TOTAL PLATE COUNT–COLOR INDICATOR                       1407
                                                                                       [Food and Drug Administration] Bacteriological Analytical Man-
                                                                                       ual (7).
                                                                                            Samplepreparation. Food samples were prepared by adding
                                                                                       225 ml of sterile Butterfield’s phosphate buffer, pH 7.2, to a 25-
                                                                                       g sample of food in a stomacher bag. Each sample was processed
                                                                                       in a Stomacher 400 (Tekmar Dohrman, Cincinnati, Ohio) at me-
                                                                                       dium speed for 1 to 2 min to break up the sample and dislodge
                                                                                       bacteria. Dilutions were prepared in Butterfield’s buffer. A mini-
                                                                                                                                         21     25
                                                                                       mumoftwoconsecutive dilutions ranging from 10       to 10   were
                                                                                       plated for each sample. Sample dilutions were chosen based on
                                                                                       the expected bacterial numbers for that food product. Foods tested
                                                                                       are listed in Tables 1 and 2.                                            Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/12/1404/1663823/0362-028x-62_12_1404.pdf by Indonesia user on 13 September 2022
                                                                                            SimPlate TPC-CI. Dehydrated medium, provided as a pre-
                 FIGURE 2. Regression analysis for aerobic plate count using           measured sterile powder by IDEXX Laboratories, was reconsti-
                 SimPlate TPC-CI versus SPC agar for 158 food samples tested           tuted with sterile diluent (deionized water or Butterfield’s buffer)
                 at IDEXX Laboratories.                                                before use. Each vial of TPC-CI powder makes 100 ml of medi-
                                                                                       um, which is enough for 10 tests. The prepared food sample was
                                                                                       added to the center landing pad of the SimPlate device followed
                 resorufin or a colorless dihydroresorufin. Variations in color          by the TPC-CI medium for a total volume of 10.0 ml (6 0.2) per
                 of positive wells, such as peach and brown, also occur.               plate (i.e., for a 1-ml sample, add 9 ml of medium). Samples from
                 Positive wells are counted and compared to a most probable            0.1 to 2 ml can be accommodated in this manner. Medium and
                 number (MPN) chart to find the MPN of the sample. The                  sample were distributed into the 84 separate wells of the SimPlate
                 results of internal (IDEXX Laboratories) and external (in-            device by gentle swirling. Excess medium was collected in the
                 dependent) validation of the method against different plate           sponge cavity located on the side of the SimPlate device by tip-
                                                                                       ping the plate to the side. SimPlates were stacked, inverted, and
                 count methods indicate that TPC-CI has equivalent sensi-              incubated for 24 h at 358C. Dairy foods are incubated at 328C.
                 tivity for foodborne bacteria but requires only half the in-          Following incubation, positive wells were counted. All color
                 cubation time. This suggests that TPC-CI is a suitable al-            changes from dark blue, including pink, purple, peach, white,
                 ternative to currently used total plate count methods.                beige, and brown, were counted as positive. Negative control
                              MATERIALSANDMETHODS                                      plates provided a reference for comparison. The MPN chart pro-
                                                                                       vided with the SimPlate device lists the number of positive wells
                      Procedure outline. Nine laboratories participated in this        and reports the MPN per plate. Confidence intervals around the
                 study. Validation at IDEXX Laboratories and three independent         MPNcalculations are available by request from the manufacturer
                 food laboratories compared SimPlate TPC-CI with SPC agar. Five        (IDEXX Laboratories). The MPN value takes into account the
                 food manufacturers tested SimPlate TPC-CI against different           excess liquid poured off into the sponge. MPN/g (ml) was cal-
                 methods, including orange serum agar (OSA), Petrifilm, Redigel,        culated by dividing the MPN/SimPlate by inoculum volume and
                 and SPC. Food samples included raw meats, fruits and vegetables,      multiplying by the reciprocal of the dilution.
                 frozen foods, dry goods including grain products and mixes, and            Methods for comparison. For internal validation, standard
                 dairy products. Procedures followed Association of Official An-        methods agar (Acumedia Mfg., Baltimore, Md.) was autoclaved
                 alytical Chemists’ Official Methods of Analysis (1), Standard          and tempered to 488C in a water bath. Samples were added to a
                 Methods for the Examination of Dairy Products (6), and the FDA        petri dish followed by the molten agar. Gentle swirling distributes
                 FIGURE 3. Regression analysis using SimPlate TPC-CI against alternative methods, SPC, OSA, Petrifilm, or Redigel for determining
                 the aerobic plate count of food for 403 data points collected from seven different laboratories.
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...Journal of food protection vol no pages copyright q international association milk and environmental sanitarians anewmediumfor determining the total plate count in c f smith d e townsend idexx laboratories inc one drive westbrook maine usa ms received april accepted july abstract downloaded from http meridian allenpress com jfp article pdf x by indonesia user on september simplate for color indicator tpc ci me is a new medium that incorporates redox dye resazurin to detect quantify bacteria enumeration achieved most probable number method using device viable are detected each well biochemical reduction which blue pink resorun or clear dihydroresorun indicators results after h incubation highly correlated with standard agar correlation coefcients studies conducted at ve ranged side comparisons against four additional test sites alternative methods aerobic reported similar comparison r slopes linear regression analysis all y intercepts ranging samples used validation included raw product...

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