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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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