TEMPO system :

AFNOR validation according to the ISO 16140 standard

Danile SOHIER1 *, Maryse RANNOU1 , Florence GORSE2
1ADRIA Dveloppement , expert lab at the AFNOR validation committee, France, daniele.sohier@adria.tm.fr / 2 BioMrieux, France

TEMPO system is a new automated RELATIVE LINEARITY STUDIES

method associating an innovative card For each alternative methods, the relative linearity was investigated
with an adapted medium to ensure by serial dilutions of pure culture mixed with 5 food types. The
rapid enumeration of several quality inoculation levels were 100 - 500 1000 - 5.103 - 5.104 CFU/g. The
indicators. It replaces serial dilutions linearity of TEMPO EC, TEMPO TVC and TEMPO TC is
and tedious plate reading with a simple Figure 1a :Tempo Reagent
characterised by linear correlation coefficients superior to 0.96; the
1/10 dilution and an automated MPN equations are shown in Table 1.
(Most Probable Number) enumeration Table 1 : TEMPO EC, TEMPO TVC, TEMPO TC linear regressions
method using 3x16 miniaturized tubes Tempo methods
(Figures 1a, 1b, 1c). Product matrices Tempo EC Tempo TVC Tempo TC
TEMPO EC, TEMPO TVC and TEMPO Dairy matrix Log Tempo EC = 0.92 log ISO + 0.47 Log Tempo TVC = 1.01 log ISO + 0.02 Log Tempo TC = 1.07 log ISO - 0.13
TC allow bacterial count of respectively
Meat matrix Log Tempo EC = 0.89 log ISO + 0.51 Log Tempo TVC = 1.01 log ISO + 0.14 Log Tempo TC = 0.98 log ISO + 0.07
E. coli, Total Viable flora and Total Figure 1b : Tempo Filler Seafood matrix Log Tempo EC = 0.13 log ISO 0.05 Log Tempo TVC = 1.05 log ISO 0.14 Log Tempo TC = 1.13 log ISO - 0.46
Coliforms. Comparison of these (three)
Vegetable matrix
alternative methods with the Log Tempo EC = 1.07 log ISO 0.03 Log Tempo TVC = 1.10 log ISO 0.28 Log Tempo TC = 1.22 log ISO - 0.72

Pet food matrix

correspondent ISO standard plate Log Tempo EC = 0.16 log ISO 0.39 Log Tempo TVC = 1.14 log ISO 0.40 Log Tempo TC = 1.05 log ISO - 0.17

count methods (NF ISO 16649-2, ISO

4833, ISO 4832) were done according RELATIVE ACCURACY STUDIES
to the ISO 16140 standard and AFNOR The relative accuracy studies were performed by testing 50 naturally
technical rules. Figure 1c : Tempo Reader contaminated food products including raw and processed meat,
poultry, dairy products, seafood, vegetables, ready -to-eat meals, and
SENSITIVITY and SPECIFICITY STUDIES pet food (Figure 3).
The sensitivity and specificity of TEMPO EC and TEMPO TC
were assessed using pure cultures of 30 targeted strains and 20 OLS 1 Regression

non-targeted strains. All TEMPO EC results were in agreement

with those obtained with NF EN ISO 16649-2 reference method,
6,00

Alternative method
TEMPO EC accuracy study
while TEMPO TC shows higher sensitivity and specificity than Contamination levels :
4,00

the ISO 4832 standard. Log 1 CFU/g to Log 4.90 CFU/g

2,00

0,00

COLLABORATIVE STUDIES
0,00 2,00 4,00 6,00

Pasteurised milk samples were contaminated with 4 different Log Tempo EC = 1.00 log ISO + 0.36 Reference method

levels of pure cultures and analysed by the reference and

alternative methods within more than 8 laboratories for each
OLS 1 Regression

method (Figure 2a, 2b, 2c and Table 2). TEMPO TVC accuracy study
8,00
Alternative method

Contamination levels :
A R A R A 6,00
0,8

0,6
Contamination levels 0,8
R A R A R A
Log 1 CFU/g to Log 6.60 CFU/g 4,00

1 000 to 10 000 2,00

0,4 100 to 1 000 0,6 Contamination levels
10 to 100 1 000 to 10 000 0,00
0,2 0,4 100 to 1000 0,00 2,00 4,00 6,00 8,00
10 to 100
Log Tempo TVC = 1.03 log ISO 0.17 Reference method
0 0,2
TEMPO EC

TEMPO TVC

TEMPO TC

Figure 2 a : Repeatability limits 0 TEMPO EC

TEMPO TVC

TEMPO TC

GMFR regression
Figure 2 b : Reproducibility limits
25.0
6 R A R A R A

5 8,000
Table 2 :
TEMPO TC accuracy study
Alternative method

Contamination levels
4 Bias between TEMPO E C, TEMPO EC, TEMPO EC 6,000
10 to 100 and the corresponding standard method
3 100 to 1 000 Tempo m e t h
ods Contamination levels :
Log 1 CFU/g to Log 6.15 CFU/g
1 000 to 10 000 4,000
Contamination levels Tempo EC Tempo TVC Tempo TC
2
10 to 100 0.02 0.10 - 0.05 2,000
1 100 to 1 000 0.14 0.09 0.08
0 TEMPO EC

1 000 to 10 000 0.31 0.20 0.02 0,000
TEMPO TVC

TEMPO TC 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00

Figure 2c: Laboratory dispersion

Log Tempo TC = 0.98 log ISO 0.25 Reference method

Depending on the contamination levels tested, EC, TEMPO

TEMPO TVC and TEMPO TC methods show similar or higher Figure 3 : TEMPO EC, TEMPO TVC and TEMPO TC accuracy
limit of repeatability and limit of reproducibility values as studies regressions, including all products
compared to standard plate count methods. TEMPO alternative
methods are characterised by a better laboratory results According to the accuracy studies, TEMPO EC, TEMPO TVC and
dispersion. The bias between both methods are non significant. TEMPO TC give results comparable to standard plate count
methods.
CONCLUSION :TEMPO system offers important economic savings by standardizing the analysis and minimizing the training time, the
volume of wastes and the number of operations. Workflow studies demonstrate a 2 to 3 fold reduction of the manipulation time using
TEMPO methods as compared to correspondent ISO reference methods.
According to these performance assessments, TEMPO EC, TEMPO TVC and TEMPO TC methods represent valuable alternative for
automated methods for E. coli, Total Viable flora and Total Coliforms enumeration in food.