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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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