TEMPO system :

AFNOR validation according to the ISO 16140 standard

Danile SOHIER1 *, Maryse RANNOU1 , Florence GORSE2

Dveloppement , expert lab at the AFNOR validation committee, France, daniele.sohier@adria.tm.fr / 2 BioMrieux, France

TEMPO system is a new automated

method associating an innovative card
with an adapted medium to ensure
rapid enumeration of several quality
indicators. It replaces serial dilutions
and tedious plate reading with a simple
1/10 dilution and an automated MPN
(Most Probable Number) enumeration
method using 3x16 miniaturized tubes
(Figures 1a, 1b, 1c).
TEMPO EC, TEMPO TVC and TEMPO
TC allow bacterial count of respectively
E. coli, Total Viable flora and Total
Coliforms. Comparison of these (three)
alternative
methods
with
the
correspondent ISO standard plate
count methods (NF ISO 16649-2, ISO
4833, ISO 4832) were done according
to the ISO 16140 standard and AFNOR
technical rules.

Figure 1a :Tempo Reagent

Table 1 : TEMPO EC, TEMPO TVC, TEMPO TC linear regressions

Tempo methods

Contamination levels

100 to 1 000

0,4

Figure 1c : Tempo Reader

0,2

0,6

Contamination levels
1 000 to 10 000
100 to 1000

0,4

10 to 100

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

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

Vegetable matrix

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

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

RELATIVE ACCURACY STUDIES

The relative accuracy studies were performed by testing 50 naturally
contaminated food products including raw and processed meat,
poultry, dairy products, seafood, vegetables, ready -to-eat meals, and
pet food (Figure 3).
OLS 1 Regression

TEMPO EC accuracy study

Contamination levels :
Log 1 CFU/g to Log 4.90 CFU/g
Log

Tempo

6,00
4,00
2,00
0,00
0,00

2,00

EC = 1.00 log ISO + 0.36

4,00

6,00

Reference method

OLS 1 Regression

TEMPO TVC accuracy study

Contamination levels :
Log 1 CFU/g to Log 6.60 CFU/g
Log

Tempo

8,00
6,00
4,00
2,00
0,00
0,00

TVC = 1.03 log ISO 0.17

2,00

4,00

6,00

8,00

Reference method

0,2

TEMPO TVC

TEMPO TC

Figure 2 a : Repeatability limits

25.0
R

TEMPO EC

Contamination levels

10 to 100
100 to 1 000
1 000 to 10 000

3
2
1

GMFR regression

TEMPO TC accuracy study

Contamination levels :
Log 1 CFU/g to Log 6.15 CFU/g

Tempo m e t h
ods
Contamination levels

100 to 1 000
1 000 to 10 000

TEMPO TC

Table 2 :
Bias between TEMPO E C, TEMPO EC, TEMPO EC
and the corresponding standard method

10 to 100

TEMPO TVC

TEMPO EC

TEMPO TVC

Figure 2 b : Reproducibility limits

Tempo EC

Tempo TVC

Tempo TC

0.02
0.14
0.31

0.10
0.09
0.20

- 0.05
0.08
0.02

TEMPO TC

8,000

Alternative method

TEMPO EC

Meat matrix

10 to 100

Log Tempo TC = 1.07 log ISO - 0.13

0,8

1 000 to 10 000

Tempo TC

Log Tempo TVC = 1.01 log ISO + 0.02

0,6

Tempo TVC

Log Tempo EC = 0.92 log ISO + 0.47

Alternative method

Figure 1b : Tempo Filler

COLLABORATIVE STUDIES
Pasteurised milk samples were contaminated with 4 different
levels of pure cultures and analysed by the reference and
alternative methods within more than 8 laboratories for each
method (Figure 2a, 2b, 2c and Table 2).
R

Tempo EC

Dairy matrix

Product matrices

SENSITIVITY and SPECIFICITY STUDIES

The sensitivity and specificity of TEMPO EC and TEMPO TC
were assessed using pure cultures of 30 targeted strains and 20
non-targeted strains. All TEMPO EC results were in agreement
with those obtained with NF EN ISO 16649-2 reference method,
while TEMPO TC shows higher sensitivity and specificity than
the ISO 4832 standard.

0,8

RELATIVE LINEARITY STUDIES

For each alternative methods, the relative linearity was investigated
by serial dilutions of pure culture mixed with 5 food types. The
inoculation levels were 100 - 500 1000 - 5.103 - 5.104 CFU/g. The
linearity of TEMPO EC, TEMPO TVC and TEMPO TC is
characterised by linear correlation coefficients superior to 0.96; the
equations are shown in Table 1.

Alternative method

1ADRIA

6,000

4,000

2,000

0,000
0,00

Figure 2c: Laboratory dispersion

TEMPO

Depending on the contamination levels tested,

EC,
TEMPO TVC and TEMPO TC methods show similar or higher
limit of repeatability and limit of reproducibility values as
compared to standard plate count methods. TEMPO alternative
methods are characterised by a better laboratory results
dispersion. The bias between both methods are non significant.

Log

Tempo

TC = 0.98 log ISO 0.25

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

Reference method

Figure 3 : TEMPO EC, TEMPO TVC and TEMPO TC accuracy

studies regressions, including all products
According to the accuracy studies, TEMPO EC, TEMPO TVC and
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.