Istituto zooprofilattico verification report and data validation

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SCHEDA RF-RELFIN1.ver1
Ministero della Salute
DIPARTIMENTO DELLA SANITA’ PUBBLICA E
DELL’INNOVAZIONE
Direzione Generale della Ricerca Sanitaria e
Biomedica e della Vigilanza sugli Enti
Ufficio III-IV
Institute/D.I. Istituto Zooprofilattico Sperimentale del Lazio e della
Toscana M. Aleandri
Direzione operativa Controllo dell’igiene, produzione e
trasformazione del latte
Title of research Development of a rapid, fast and low-cost analytical kit to
evaluate quality and safety of milk and dairy products
G R-2011-02348227
Start date 1/12/2014 End date 30/11/2017
Final Relation
A. Research partecipants:
1) IZSLT - Istituto Zooprofilattico Sperimentale del Lazio e della Toscana
2) INBB - Istituto Nazionale Biostrutture e Biosistemi - Consorzio Interuniversitario
3) IZSAM - Istituto Zooprofilattico Sperimentale Abruzzo e Molise G. Caporale
B. Objectives of the project:
The aim of this project is the validation of a quick, sensitive and cheap kit for quality
control and microbiological safety of milk and dairy products. In particular, the research object is the
MBS colorimetric method, developed by MBS Srl and Roma Tre University, for rapid execution of
microbiological analyses. These reliable analyses can be performed by unskilled personnel, in
absence of any laboratory equipment, quickly and with reduced costs.

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Ufficio III-IV
C. Methodology applied:
Validation experiments of the MBS method were carried out in Istituto Zooprofilattico
Sperimentale del Lazio e della Toscana (IZSLT) “M. Aleandri” – “Direzione operativa Controllo
dell’igiene, produzione e trasformazione del latte”.
Materials and methods:
The determination of the Total Bacterial Count at 30°C was carried out on 186 samples of raw cow’s
milk in according to ISO 4833-1: 2013 Microbiology of the food chain -- Horizontal method for the
enumeration of microorganisms -- Part 1: Colony count at 30 degrees C by the pour plate technique.
The reference method is indicated in COMMISSION REGULATION (EC) No 1664/2006 of 6
November 2006 amending Regulation (EC) No 2074/2005 as regards implementing measures for
certain products of animal origin intended for human consumption and repealing certain
implementing measures.
For the criteria of verification in Annex III, Section IX, Chapter I, Part III to Regulation (EC) No
853/2004 the following rules should be applied as reference methods:
a) EN/ISO 4833 which specifies a method for enumeration of microorganisms that are able to grow
and form colonies in a solid medium after aerobic incubation at 30 °C.
Operating procedures:
1 ml of each sample of milk and serial decimal dilutions were analyzed using the Puor plate method
on Milk agar. Inoculated plates were incubated at 30°C for 72 hours. After the fixed time, the count of
colonies was made. All samples were analyzed in duplicate.
In parallel, the Total Bacterial Count at 30°C was also carried out using the MBS method on the same
samples of milk. The MBS method was performed using TVC (Total Viable Count) vials for the
quantification of Total Bacterial Count. All vials were produced by MBS srl, Rome, Italy. All samples
were analyzed in duplicate. The TVC vials were filled with 10 ml of sterile distilled water and 1 ml of
water samples was added to each vial. After inoculation, vials were incubated for a maximum 30
hours at 30 °C in a in the MBS Multireader. This device is a thermostated colorimeter that

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Ufficio III-IV
automatically looks at the color change of vials and calculates the bacterial concentration in the
sample. The starting color for TVC vials is blue. In the presence of microorganisms, the vials’color
changes to yellow, indicating a positive result. The persistence of the initial color after 30 hours
indicates the absence of tested microorganisms, and consequently a negative result.
- The duplicate tests performed using the reference method were subjected to verification of the
proportionality of the counts.
- The results of the counts of the Total Bacterial Count, obtained using the reference method, were
subjected to verification of the degree of agreement with the theoretical distribution model.
For the reading, plates with a number of colonies included between 10 and 300 were considered.
The expressions used for this verification are shown below.
Linearity was assessed using the following formula:
ci Number of colonies detected in the plate
Ri Relative quantity of sample inoculated to the dilution under test
Results are proportional if the following expression is satisfied:
𝐺𝐺𝑚𝑚−1
2
≤ 𝜒𝜒𝑔𝑔.𝑙𝑙
2
con g.l. = n. - 1
N.B. Samples with critical and unacceptable values were not included in the regression calculation.
The distribution in classes of CFU/ml was as follows:
Classes of CFU/ml Number of samples %
< 100.000 104 55,9
100.000 - 500.000 51 27,4
500.000 - 1.000.000 15 8,1
>1.000.000 16 8,6
The results obtained using the reference method were transformed into Log10 to make the normal

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distribution.
A comparison test of the determinations of the Total Bacterial Count using the reference method vs
MBS method was also performed, replicating 10 times the parallel test for each method on two
samples of bovine raw milk corresponding to two levels of bacterial load: 104
CFU/ml e 106
CFU/ml.
At the Istituto Zooprofilattico Sperimentale Abruzzo e Molise G. Caporale (IZSAM) further tests
were carried out comparing the MBS method with the reference method of plate counting. For each
day of analysis in laboratory a bottle of cow's milk was kept under stirring on a magnetic plate.
Subsequently the contents of the bottle were divided into four sterile containers. Each container, in
turn, was divided into two, in order to get to have 8 samples for each day of analysis.
During the analysis, the samples were kept at 4°C±2°C to avoid an increase in charge.
Analysis performed:
The quantification of the Total Viable Count was performed according to the procedure
IZSTEB3.1.2SOP061 which has, as its reference, the Decreto Ministeriale 26 Marzo 1992: Attuazione
della decisione n. 91/180/CE, pubblicato sulla G.U. n. 90 del 16 Aprile del 1992, concerning the set
up of methods of analysis and testing relating to raw milk and heat-treated milk.
For each day of analysis a sterility test of the agar medium used was set up, at 21 ° C and 37 ° C, to
ensure the control of the analysis process according to the IZSTE procedure B3.1.3 SOP005.
Parallel to the reference method of plate count, the analysis of samples was performed using the MBS
method as indicated above for IZSLT.
D. Results:
The MBS reagent for the quantification of Total Bacterial Count was validated through the
analysis of different milk stocks. This validation was performed through a comparison with the
reference method on naturally contaminated samples. In particular, the following parameters were
analyzed:
- Accuracy, assessed by measuring:
a) repeatability (precision of multiple determinations of a single sample in a single test);
b) inter-assay variability (precision for multiple determinations of a single sample, controls

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and reagents analyzed in different tests);
c) reproducibility (precision between the laboratories involved in the project).
- Specificity, defined as the ability of the method to measure the analyte with precision and
specificity, even in the presence of other components possibly present in the sample matrix.
- Linearity, defined as the ability of the analytical procedure to produce results that are
directly proportional to the concentration (quantity) of the analyte itself. The detection limit (LOD),
defined as the lowest level of analyte that can be detected, will also be evaluated.
- Precision, defined as the correspondence of the results obtained from the procedure
examined with respect to the real value.
In particular, at the IZSLT, the results of the averages of the Total Bacterial Count, determined with
the reference method of plate count, were compared with the averages of the results obtained with
the MBS method for the same 186 samples of bovine raw milk. The regression line of the decimal
logarithms of the Total Bacterial Count (expressed as CFU/mL) obtained using the reference method
was then determined in relation to the time for color change (expressed as hour) obtained by the
MBS method (Fig. 1).

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Figure 1: Regression line of the decimal logarithms of the Total Bacterial Count (expressed as
CFU/mL) obtained using the reference method in relation to the time for color change (expressed as
hour) obtained by the MBS method .
Through this regression, a correlation of R2
= 0,61 (r = 0,78) was carried out.
The relation to be adopted for the calculation of the bacterial load (expressed as CFU/ml) estimated by
the time of color change for the MBS method is given by the following equation:
Log CFU/ml = - 0,2522 * MBS time + 7,2815
Subsequently, the determination of the residuals of the previously calculated linear regression was
determined (Fig 2).
Figure 2: Determination of residues of linear regression.

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The distribution of residues obtained through the use of the above-mentioned equation shows a normal
pattern.
Test di Bland-Altman
The results obtained with the two methods were also graphically compared by the Bland-Altman test
(Fig. 3).
Figure 3: Test di Bland-Altman. The mean values of the two methods are shown on the X axis. The
difference of the two values is shown on the Y axis.
This allowed us to determine the confidence interval (CI) at 95% of the mean difference:
𝐼𝐼𝐼𝐼 = Mean difference ±
1.96 ∗ 𝑠𝑠
√𝑛𝑛
s = standard deviation of differences
n = N° of samples

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Lower limit
IC = 0,000057 - 1,96 * 0,49 = - 0,951
Upper limit
IC = 0,000057 + 1,96 * 0,49 = 0,951
The results obtained with the Bland-Altman Test compared to the limits of the confidence interval
obtained show a substantial reliability with respect to the interchangeability of the two methods.
Comparison of methods
The relation adopted for the assessment of the comparison of results, obtained using both the methods,
was the following:
A = Average of results obtained using the reference method
B = Average of results obtained using the MBS method
The results obtained were as follows:
First level (bacterial load: 104
CFU/ml):
Second level (bacterial load: 104
CFU/ml):
The comparison between the two averages of results, obtained with the two methods, provides, for

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both the considered levels, a value according to the considered relation.
At the IZSAM, the results of the averages of the Total Bacterial Count, determined with the reference
method of plate count, were compared with the averages of the results obtained with the MBS method
for the same 104 samples of bovine raw milk.
The regression line of the decimal logarithms of the Total Bacterial Count (expressed as CFU/mL)
obtained using the reference method was then determined in relation to the time for color change
(expressed as hour) obtained by the MBS method (Fig. 4).
Figure 4: Regression line of the decimal logarithms of the Total Bacterial Count (expressed as
CFU/mL) obtained using the reference method in relation to the time for color change (expressed as
hour) obtained by the MBS method.
As indicated in Fig. 4, a correlation of R2
= 0,66 was carried out.
The relation to be adopted for the calculation of the bacterial load (expressed as CFU/ml) estimated by
y = -0,313x + 8,430
R² = 0,661
0
1
2
3
4
5
6
7
8
9
0 2 4 6 8 10 12 14
LogCFU/mlcontasupiastra
Viraggio fiale MBS (ore)

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Ufficio III-IV
the time of color change for the MBS method is given by the following equation:
Log CFU/ml = - 0,3134 * MBS time + 8,4307
Subsequently, the determination of the residuals of the previously calculated linear regression was
determined (Fig 5).
Figure 5: Determination of residues of linear regression.
The distribution of residues obtained through the use of the above-mentioned equation shows a normal
pattern.
Test di Bland-Altman
The results obtained with the two methods were also graphically compared by the Bland-Altman test,
(Fig. 6). Even in this case, the results obtained with the Bland-Altman test support the correspondence
between the bacterial concentration values obtained using the reference method and the MBS method,
further supporting the interchangeability of the two methods.
-1,5
-1
-0,5
0
0,5
1
1,5
2
2,5
3
0 2 4 6 8 10 12 14

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Figure 6: Test di Bland-Altman. The mean values of the two methods are shown on the X axis. The
difference of the two values is shown on Y axis.

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E. Abstract
The results obtained in collaboration with the IZSLT and IZSAM reference centers using
the MBS method demonstrate the accuracy of this method for the determination of the Total Bacteria
Count in the raw cow's milk matrix. The results obtained with the MBS method are, in fact,
comparable with those obtained with the reference method of the plate count. These data highlight
the potential of the MBS method as a valid tool for reliable microbiological analysis within the agro-
food sector. Its characteristics of reproducibility, accuracy, economy and portability make it
particularly suitable for self-control by small and medium-sized dairy companies, as it ensures to
control autonomously the microbiological quality and safety along the entire food supply chain even
in the absence of an internal microbiological laboratory.

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