Evaluation of a New Automated Method for Lactic Acid Bacteria Enumeration in Food Products Using the TEMPO® System
Grégory Devulder, Cidem Ilter, Audrey Maingue.
RESULTS
INTRODUCTION
1/10 dilution
Decimal
dilutions
DILUTIONS
Result agreement for within range between TEMPO and the reference method
1mL
Pour ~15 mL MRS (De Man,
Rogosa and Sharpe)
Analysis:
The comparison in terms of enumeration (T-test, bias and regression) was performed on
within range data: this means that for both methods, the log decimal transformation is
possible. The analysis of below and above range was performed using the rate of
agreement results.
Distribution of food products tested by category:
# Matrices tested at
35°C
Sample Type
Bakery
Miscellaneous *
17
Dairy
53
Poultry
Meat
83
Prepared food
Environment
9
Vegetables / Cereals
Fish and seafood
60
Number of generated results
* Delicatessen, Pet food, Dressing, etc…
# Matrices tested at
35°C
96
28
52
54
1276 (452 products)
PREPARATION
# data
% agreement
Within range
451
91.1 (411)
Below/above range
805
98.1 (790)
1256
95.6 (1201)
INCUBATION
MATERIALS AND METHODS
40-48h - 35°C - aerobic conditions
72h - 35°C - anaerobic conditions
READING
Total
The bias (mean of the log difference)
is -0.11 [-0.05 ; -0.16] with a
confidence interval at 95% close to 0.
1256 within range and below above range results were used for the comparison. We
observed a rate of agreement of 91.1% and 98.1% for within range and below above range
respectively and a global rate of agreement higher than 95% on all food categories.
No significant difference was observed regarding the different categories of food products
tested.
Log TEMPO vs Log Reference
The TEMPO LAB method presents important advantages compared to the reference method:
The enumeration of Lactic Acid Bacteria is often fastidious and time consuming due to the high number of
dilutions. The use of the TEMPO system allows an enumeration range of 3.7 log with the same card which decreases
significantly the number of dilutions: one card for 3 plates.
The incubation of TEMPO card is performed aerobically compared to the MRS method or PetrifilmTM which are
incubated in anaerobic conditions.
The incubation time is 40-48h compared to 72 hours for the reference method allowing time saving for the result
and space saving in the incubator.
CONCLUSION
Results obtained with the TEMPO LAB parameter were not statistically significantly different
than those obtained by plate count on MRS agar, showing a satisfactory agreement between
both methods. TEMPO LAB method can be considered as an effective rapid alternative method
for the enumeration of lactic acid bacteria in food products offering significant advantages to
the reference method.
8
DISCUSSION
6
The linear regression :
y=0.22 + 0.93x
shows a coefficient of correlation
of 0.94.
4
At 35°C, TEMPO LAB shows equivalent results to the Compendium method with a global rate of agreement close to
96%. A slight tendency for under enumeration is observed on all food categories. Some under enumeration could
explained by the presence of yeasts on MRS. It is sometimes recommended to add sorbic acid to the MRS medium if
there is a risk of yeast contamination (ex ISO 15214). The performance are comparable or better than performance
observed for the comparison between PetrifilmTM and the reference method which shows a slight over enumeration.
Log (TEMPO LAB)
Reference method:
Rehydrated MRS medium was prepared according to manufacturer’s instructions. The pH
of the medium was adjusted to 5.5 after autoclaving using sterile glacial acetic acid to
obtain the pH-modified agar. Inoculated plates in duplicate were incubated anaerobically at
35°C for 72 ± 3 hours.
TEMPO method:
TEMPO LAB is a method for Lactic Acid Bacteria enumeration, based on the familiar
format of the TEMPO MPN procedure. The method uses a new selective dehydrated
culture medium and an enumeration card containing 48 wells across 3 different dilutions
for the automatic determination of the MPN. Filled cards were incubated aerobically at
35°C for 40-48 hours.
Alternative method (PetrifilmTM):
PetrifilmTM Method was performed according to manufacturer’s instructions. The MRS
broth used as diluent is Difco Lactobacilli MRS broth 288130, pH 6.5 adjusted to 5,7 after
autoclaving with sterile glacial acetic acid. Inoculated Aerobic Count Plate were incubated
anaerobically at 35°C for 48 ± 3 hours.
On 1276 results (452 different matrices),
• 18 eliminated reference results due to incoherence between successive dilutions.
• 2 eliminated TEMPO results due to difficulties for the system to interpret the card
2
In this study, we compared the new TEMPO LAB enumeration method to the
Compendium method for the microbiological examination of foods: MRS agar incubated at
35°C anaerobically for 72 ± 3 hours. At the same time, the 3MTM PetrifilmTM method was
compared to the reference method on a subset of data.
1 mL or 0.1 mL
0
Whereas Lactic Acid Bacteria (LAB) are considered beneficial in the manufacture of
fermented products they can also act as spoilage organisms, which shorten product shelf
life and degrade product quality. Traditional methods for enumerating LAB in food
products involve time-consuming plating techniques.
Sample Type
P3-46
bioMérieux SA, Marcy-l'Etoile, France
0
2
4
6
8
Log (Reference)
Result agreement for within range between PetrifilmTM and the reference method
The same analysis was performed on 216 food products to compare PetrifilmTM and the
reference method:
# data
% agreement
Within range
102
84.3 (86)
Below/above range
114
93.0 (106)
216
88.9 (192)
Total
The bias (mean of the log difference)
is +0.24 [0.07 ; - 0.41] with a
confidence interval at 95% close to 0.