TEMPO STA AOAC ILM article, Sept/Oct 2010 - bioMerieux

A O A C R E S E A R C h I N S T I T u T E N E w S
bioMérieux TEMPO ® STA Test Granted
PTM Status
bioMérieux Inc.
TEMPO ® STA (Staphylococcus
aureus) Test
Cat. No.: 80002
PTM Status: December 24, 2009
Certificate No.: 120901
Summary of Validation Claims
The TEMPO ® STA
(Staphylococcus aureus)
method is an automated
system for the enumeration of
S. aureus. The method uses a
selective dehydrated culture
medium and an enumeration
card containing 48 wells across
three different dilutions for the
automatic determination of the
most probable number (MPN).
TEMPO STA was validated
and certified as a Performance
Tested Method SM (PTM) by the
AOAC Research Institute (RI)
as an effective method for the
enumeration of S. aureus in
a variety of foods, including
meat, poultry, dairy, seafood,
egg, confectionary, and
vegetables.
The TEMPO STA method
was compared to AOAC Official
Method of Analysis SM (OMA)
975.55, which included confirmation.
A combination of the
difference in log 10 distributions
and t-tests at the 5% level
were used to analyze the data
and compare performances.
The TEMPO method was shown
to be statistically equivalent to
the reference method in both
the internal and independent
food studies. The TEMPO
STA reagents were stable and
provided highly reproducible
results when compared
between lots. Assay ruggedness
was demonstrated for two critical
parameters, time of incubation of the
test and incubation temperature of
the test.
Figure 1. TEMPO STA test reagents.
Figure 2. TEMPO Preparation station for inoculation, with distribution
of a diluted food sample in culture media, then filling and sealing of
TEMPO cards using the TEMPO Filler.
Figure 3. TEMPO Reading station for reading and interpretation of
TEMPO cards using the TEMPO Reader.
principle of the Test
The TEMPO STA test consists of a
vial of culture medium and a card, which
are specific to this test (Figure 1). The
culture medium is inoculated with the
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sample to be tested. The inoculated
medium is transferred
by the TEMPO Filler into
the card containing 48 wells
of three different volumes
(Figure 2). The card contains
three sets of 16 wells (small,
medium, and large wells) with
a 1 log difference in volume
for each set of wells. The
card is designed to simulate
the MPN method. The card
is then hermetically sealed
in order to avoid any risk of
contamination during subsequent
handling. The culture
medium contains a fluorescent
pH indicator, which, when
its pH is neutral, emits a
signal detected by the TEMPO
Reader. S. aureus present in
the card assimilate the nutrients
in the culture medium
during incubation, resulting
in a decrease in pH and the
extinction of the fluorescent
signal. After incubation, the
cards are transferred to an
automated card reader that
detects the fluorescent signal
(Figure 3). Depending on the
number and type of the positive
wells in the 3 log dilution
range, the TEMPO system
calculates the number of
S. aureus present in the
original sample according to
a calculation based on the
MPN method.
Summary of pTM Validation
TEMPO STA was validated
according to AOAC RI PTM
guidelines. The validation
consisted of two main parts:
internal and independent testing.
Internal testing included
inclusivity and exclusivity testing as well
as ruggedness, stability, and lot-to-lot
studies. Additionally, the internal testing
(Continued on page 32.)
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A O A C R E S E A R C h I N S T I T u T E N E w S
bioMérieux TEMpO ® STA Test Granted pTM Status
Continued from page 31.
included method comparison studies for
15 claimed matrixes: fresh ground beef
patties, raw milk, filet of beef, cheddar
cheese, prosciutto, vanilla ice cream,
nonfat dry milk, cooked diced chicken,
chicken salad, chocolate éclairs, tuna
salad, frozen hash browns, smoked
salmon, frozen mixed vegetables, and
frozen egg omelets. Independent laboratory
testing included method comparison
studies for three claimed matrixes,
including fresh ground beef, cooked diced
chicken, and vanilla ice cream.
Twenty test portions of each food
matrix were analyzed by both methods
and included five replicates of three
different lots that were artificially contaminated
at low, medium, and high
levels with S. aureus and five uninoculated
control samples for each food.
A 1:10 dilution of each sample was
prepared and stomached for 2 minutes.
For each diluted and stomached
sample, 1.0 mL of diluted food sample
was added to a TEMPO medium vial
that had been reconstituted with 3.0 mL
sterile distilled water. The inoculated
medium in the vial was then transferred
and sealed into the STA card by the
automated TEMPO filler. The inoculated
cards were incubated for 24 to
27 hours at 35 ± 1°C. Cards were read
using the automated TEMPO reader.
Standard method testing was performed
as detailed in OMA 975.55.
Figure 4. Regression analysis.
TAblE 1. ObSErvED biAS bETwEEN ThE TEMPO STA AND rEfErENCE METhOD
Results
The bacterial counts for TEMPO and
the reference method were transformed
to log 10 and the bias and corresponding
95% confidence interval of the bias
were calculated. Regression analysis
was also performed on the data set
along with a t-test of the mean values
for the TEMPO and the reference
method. The bias, 95% confidence
interval, and t-test results for the data
set are detailed in Table 1.
The overall percent agreement
between the TEMPO STA and reference
method was calculated by determining
the number of data points that exhibited
log differences of less than 1. The agreement
within 1 log for this study was 98%.
The mean log counts for the TEMPO STA
method on the entire data set were not
significantly different from those of the
reference method. Regression analysis of
this data set is detailed in Figure 4. The
two methods gave similar results with a
correlation coefficient (R) 0.91, intercept
–0.018, and slope 0.99. The analysis
of bias yielded a confidence interval
result that indicates that there is no bias
between the TEMPO STA and the reference
method for these study data.
bias Confidence interval P value
TEMPO STA vs reference –0.047 [–0.11; 0.01] 0.115
Independent Laboratory
The results of this independent
evaluation demonstrate that the bio-
Mérieux TEMPO STA method is a rapid,
reliable method for the enumeration of
S. aureus. The amount of time to results
is 24 hours compared to 3 to 4 days for
AOAC 975.55. The simple rehydration
of the TEMPO STA medium and addition
of sample and the elimination of serial
dilutions and pour plating significantly
reduced the sample analysis time. In
addition, the automated feature of the
TEMPO STA method eliminates the
often problematic issue of subjectivity
observed with plate count methods.
Conclusions
In the AOAC PTM validation study,
TEMPO STA method performance was
equivalent to the reference method
evaluated. By eliminating media
preparation, serial dilutions, manual
counting, and visual interpretation of
results, automation of the TEMPO STA
system provides reliable, rapid results
and significant economic savings in
labor and increased efficiency. ■
—Ronald L. Johnson, John C. Mills,
and Judith Colón-Reveles
Contributing Writers
ron.johnson@biomerieux.com
john.mills@biomerieux.com
judith.colon-reveles@biomerieux.com
TEMPO ® STA (Staphylococcus aureus) Test and
system are available worldwide. For more information,
visit www.bioMerieux.com and select
the country of interest or type in TEMPO at the
search option.
Mention of trade names or commercial products
is for identification only and does not constitute
preference over similar ones not mentioned.
If you are interested in submitting an article
regarding a test kit that has been granted
Performance Tested Method SM status, contact
Zerlinde Johnson at zjohnson@aoac.org.
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