Journal of Dairy Research (2013) 80 475–484.

© Proprietors of Journal of Dairy Research 2013 475

doi:10.1017/S0022029913000423

Effect of storage and preservation of milk samples on the response

of microbial inhibitor tests
Milagro Borràs Llopis1*, Marta Roca Marugón2, Rafael Lisandro Althaus 3 and Maria Pilar Molina Pons1
1
Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Camino de Vera, s/n 46022 Valencia, Spain
2
Centro Superior de Investigación en Salud Pública, Avd. Cataluña, 21 46020 Valencia, Spain
3
Cátedra de Biofísica, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral – R.P.L., Kreder 2805, 3080 Esperanza, Argentina

Received 17 January 2013; accepted for publication 15 July 2013; first published online 9 October 2013

The aim of this study was to evaluate the effect of storage time (4 °C) on milk samples and the use of
azidiol as preservative on the results of microbial inhibitor tests used to detect antimicrobials in milk.
For this purpose, 16 milk bulk samples divided into two aliquots, preservative-free and with azidiol,
spiked with 12 concentrations of amoxicillin, ampicillin, penicillin G and oxytetracycline, were
used. The milk samples were analysed using the BRT MRL, Delvotest MCS Accelerator and Eclipse
100 at 0, 24, 48 and 72 h stored at 4 °C. The logistic regression model was applied to study the effect
of storage time (ST), preservative (P) and their interaction (ST × P). At a concentration equivalent to the
Detection Limit (DL), the positive results of microbial inhibitor tests do not remain stable during
storage time. These results are more reproducible if samples are stored with a preservative. At
Maximum Residue Limits (MRLs) concentration microbial inhibitor tests can detect penicillin for up
to 72 h of storage. For oxytetracycline, the BRT MRL and Delvotest MCS tests presents DL exceeding
the MRL, therefore they are not sensitivity to this tetracycline (0% positive results). By contrast Eclipse
100, whose DL is lower than the MRL, gave 100% positive results during the 72 h storage period. It
can be concluded that it would be convenient to store milk samples with azidiol at 4 °C and to carry
out analyses within the first 48 h since milk sampling.

Keywords: Microbial inhibitor tests, storage, azidiol, antibiotics.

The presence of antimicrobial residues in milk supplies may the test microorganism grows and a colour change in the
have public health implications, such as the development of indicator takes place. These tests are widely utilised for
allergic reactions, interference in the intestinal flora and the the screening of inhibitors in milk, as they are quick, easy to
development of antibiotic resistance (Alanis, 2005; Demoly use and economical (Toldrá & Reig, 2006; IDF, 2010). In
& Romano, 2005). Moreover, it can interfere with the manu- addition, they have the sensitivity to detect β-lactams and
facture of dairy products (Babu et al. 1989; Berruga et al. sulphonamides, but they are not sensitive enough to detect
2007). many other antibiotics such as tetracyclines, quinolones,
To prevent these problems, the European Union macrolides and aminoglycosides (Le Breton et al. 2007;
(Regulation, 853/2004/EC) specified that raw milk intended Kantiani et al. 2009; Navrátilova, 2009; Perme et al. 2010).
for human consumption must not contain antimicrobial resi- Moreover, accuracy in milk inhibitor analysis is very im-
dues exceeding the Maximum Residue Limits (MRLs) as portant to most dairy farmers and to the dairy industry. Many
established by Regulation (2010/37/EC). factors can affect the response of these tests, such as milk
Different microbial inhibitor tests have been developed composition, somatic cell count and total bacteria count
for the detection of these residues in milk. The principle of (Althaus et al. 2003; Kang et al. 2005; Comunian et al. 2010).
these methods is based on the inhibition of the growth of the Also, other important factors can affect the results namely
Geobacillus stearothermophilus subsp. calidolactis spore. adequate milk sampling as well as storage before analysis in
This inhibition of growth is demonstrable as the colour of the control laboratories.
culture medium remains unchanged. In inhibitor-free milk, In this sense, the AOAC (2000) and the International Dairy
Federation (IDF, 1995) laid down requirements for the milk
sampling process, storage and the transport of samples. For
this purpose, storage of the samples should be such that the
*For correspondence; e-mail: [email protected] state of the sample at the time of sampling is not adversely
476 M Borràs and others

affected to any considerable extent. During transport, Table 1. Quality parameters of cow’s milk samples (n = 16)
precautions should be taken to prevent exposure to odours,
Standard
direct sunlight and other adverse conditions. The storage Parameters Mean deviation Minimum Maximum
temperature for raw milk should be between 0
4 °C, which
should be reached as quickly as possible. Also, samples must Fat (%) 3·86 0·99 3·10 4·82
Protein (%) 3·30 0·42 2·99 3·72
be dispatched immediately after sampling to the testing
Lactose (%) 4·75 0·29 4·18 5·25
laboratory.
Non-fat solids (%) 8·83 0·43 8·29 9·35
Furthermore, some authors suggest a possible decrease SCC (× 103 cel/ml) 150 316 12 400
in concentration and/or the loss of antimicrobial activity Bacterial count 40 12 10 60
in some molecules during the storage of milk samples in (× 103 cfu/ml)
refrigeration, for example, as in the case of penicillin G,
SCC: Somatic Cell Count
ampicillin, amoxicillin (Schenck & Friedman, 2000) and
oxytetracycline (Himanish et al. 2008; Roca et al. 2008).
Therefore, the storage time of milk samples at low temp- Hillerød, Denmark) and bacteriological count (BactoScan
eratures may influence the results of microbial inhibitor FC. Foss, Hillerød, Denmark) were analysed. Table 1 pres-
tests. ents the mean values of the composition, somatic cell counts
Also, the AOAC and IDF have established the possibility of and bacteriological counts of the milk samples.
using preservatives in milk samples (for example, bronopol The 16 milk pool samples were divided into two aliquots
or azidiol) to prevent spoilage during transport from the (n = 32) to analyse the effect of the preservative (16 pre-
producer to the laboratory, so long as their nature does not servative-free and 16 with azidiol). To prepare the azidiol
interfere with subsequent analyses. Some authors have solution and its dosage, the rules laid down by Spanish
studied the effects of the use of preservatives on the different regulations (BOE, 1728/2007) were followed. The azidiol
characteristics of milk such as composition (Sánchez et al. composition was 0·75 g chloramphenicol, 10 ml ethanol
2005; Butler & Stergiadis, 2011), somatic cell (Martínez et al. (96%), 18 g sodium azide, 45 g trisodium citrate and 0·35 g
2003; Sánchez et al. 2005) and total bacteria count bromophenol blue, mixed in 1000 ml of distilled water. The
(Elizondo et al. 2007; Sierra et al. 2009a) dose was chosen to reach a final concentration of 3·3 ml/l
In recent years, the use of azidiol has become compulsory milk.
in all milk-testing laboratories in Spain. This preservative
contains a mixture of chloramphenicol, sodium azide,
Antibiotics and spiked milk samples
bromophenol blue and sodium citrate. Chloramphenicol is
a bacteriostatic antibiotic that can affect microbial inhibitor In order to calculate the dose-response curve (IDF, 2002),
tests, as these tests are based on the inhibition of microbial each of the 32 aliquots of milk (16 preservative-free and
growth of specific microorganisms, giving ‘false positive’ 16 with azidiol) were divided in 12 aliquot milk samples,
results (Molina et al. 2003; Stead et al. 2008). for fortification with 12 different antibiotic concentrations
The objective of this study was to evaluate the effect of which in turn, were analysed 8 times by the screening
storage time at 4 °C and the use of azidiol in milk samples on methods.
the results of microbial inhibitor tests used in milk-testing The antibiotics selected for this study are widely used in
laboratories. the veterinary treatment of cattle and were supplied by
Sigma-Aldrich (Madrid, Spain): amoxicillin (AMO, A-8523),
ampicillin (AMP, A-9518), penicillin G (PEN, Pen-Na) and
Material and methods oxytetracycline (OXY, O-5750). The concentrations of anti-
biotics varied according to the drug and method, shown
Preparation of milk samples
in Table 2.
The antimicrobial-free milk samples (‘negative milk’) used The drugs used to prepare antimicrobial solutions were
were obtained according to ISO/FDIS 13969 (IDF, 2002); handled according to the manufacturers’ instructions. To
requirements. The animals received no pharmacological avoid possible instability, all the dilutions were prepared in
treatment throughout the sampling period. The cows were in 10 ml volumetric flasks when analyses were carried out.
mid-lactation (between 60 and 200 d after calving). The Spiked milk samples were prepared from the respective stock
clinical and subclinical health status of the animals was solution in a single step (IDF, 2010).
good, with fewer than 150 000 somatic cells/ml, and the Immediately after preparation, the spiked milk samples
total viable count was below 104 cfu/ml in milk. The milk of were divided into 4 aliquots (10 ml) to evaluate the effect of
seven cows was combined to overcome individual vari- storage time at 4 °C (0, 24, 48 and 72 h).
ations in milk composition to prepare a pool sample
(1000 ml). Sixteen negative milk samples were used for the
Microbial inhibitor test
analysis of each antibiotic.
Gross composition (MilkoScan FT-120. Foss, Hillerød, The microbial inhibitor tests used were Brilliant Black
Denmark), somatic cell count (Fossomatic 5000. Foss, Reduction Test MRL (BRT MRL, AiM Analytik in Milch
 Milk storage and preservation, the response of microbial inhibitor tests 477

Produktions-und Vertriebs-GmbH, Munich, Germany),

3·75
5·25

3·75

2·55

500
450
260
4·5
4 Delvotest MCS Accelerator (Delvotest MCS, DSM Food

4
Specialties, Delft, the Netherlands) and Eclipse 100 (Eclipse,
Zeu-Inmunotec, Zaragoza, Spain), according to each manu-
facturers’ instructions. For the ‘positive control’, milk sam-
3·50
3·50

2·75

450
425
240
3·5
1·8
2·5
3·5
ples were spiked with 4 μg/kg of penicillin G. Afterwards,
5

the BRT MRL and Delvotest MCS tests were incubated in a

water bath at 64 ± 1 °C for approximately 3 and 2 h 30 min,
3·25

3·75

2·25

400
400
220
respectively. Eclipse 100 was used with a 1-h diffusion time
4·5
2·5

1·6
3

3
at room temperature and was then incubated for approxi-
mately 2 h 30 min in a water bath at 64 ± 1 °C. Visual
interpretation was carried out by three trained technicians
2·25

2·75

2·75
350
375
200
2·5

3·5

1·5

and was evaluated as ‘negative’ (yellow) or ‘positive’ (blue).

3
4

2
2·75

3·25

1·75

Statistical analysis
300
350
180
3·5

2·5
1·4

2·5
2

To evaluate the effects of storage time (ST), the preservative

Concentrations (μg/kg)

(P) and their interaction (ST × P) on the responses of the

microbial inhibitor tests, the logistic regression model used
1·75

3·25
1·50

2·25

2·25
250
325
160
2·5

1·3
1·5

was:
3

Lijkl ¼ log it½Pijkl 

1·50
2·25

1·25
2·75

1·25

¼ β0 þ β1 Ci þ β2 STj þ β3 Pk þ β23 STj  Pk þ εijkl

225
300
140
1·2
3

where: Lijkl: logit model; [Pijkl]: probability for the response

category (positive or negative); β0: intercept; β1, β2 and β3:
1·25

2·75

1·75

1·75

the parameters estimated for the model; Ci: effect of con-

200
275
120
2·5

AMO: amoxicillin; AMP: ampicillin; PEN: penicillin G; OXY: oxytetracyclin; MRL: Maximum Residue Limit
2

1
1

centration (n = 12); STj: effect of storage time (n = 4); Pk:

effect of preservative, on the dummy variable (A = 1 no
azidiol; A = 0 azidiol); STj × Pk: effect of interaction storage
1·75

0·75

0·75

150
250
100
2·5

1·5
0·8

1·5

time × preservative and εijk: residual error.

1

From the equation obtained with the logistic regression for

Table 2. Antibiotic concentrations used for the different microbial inhibitor tests

each method and antibiotic, a detection limit (DL) was deter-

mined, using preservative-free milk samples and immedi-
0·75
1·25
2·25
0·50
1·75
1·25

1·25
100
225
0·6
0·5

80

ately after preparation (0 h). The DL was calculated as the

concentration that produces 95% positive results (ISO/FDIS
13969:2002).
0·25

0·25

200

Furthermore, by using this equation at a concentration

0·5

1·5

0·4

50

60
1
2

equivalent to the DL and MRL, the positive results of micro-

bial inhibitor tests at different storage times (0, 24, 48 and
72 h) were calculated.
0
0
0
0
0
0
0
0
0
0
0
0
Delvotest MCS

Delvotest MCS

Delvotest MCS

Delvotest MCS

Results and discussion

Eclipse 100

Eclipse 100

Eclipse 100

Eclipse 100
BRT MRL

BRT MRL

BRT MRL

BRT MRL

Table 3 shows the equations used to predict the positive

Method

results of the microbial inhibitor tests and the significant

parameters in the statistical analysis (P < 0·05). An increase in
the β1 coefficient value indicates that higher antibiotic
concentrations increase the frequency of the positive results
(μg/kg)

of the methods. The β2 coefficient related to storage time

MRL

100
4

having a negative sign, indicates that the positive results

decrease when the storage time is prolonged. Parameter β3
stands for the effect of the preservative on the dose-response
Antibiotics

curve. This coefficient is only significant (P < 0·05) for the

AMO

results of the BRT MRL method with ampicillin and

AMP

OXY
PEN

oxytetracycline. Thus, the results of this test differ

478 M Borràs and others

Table 3. Logistic equation of antibiotics dose-response curves in milk analysed by microbial inhibitor tests
Goodness-of-fit test

Antibiotics Method Logist [P] = β0 + β1Ci + β2 STj + β3 Pk + β23 STi × Pk χ2 P

AMO BRT MRL L =
5·89 + 3·21C
0·03ST
0·09ST × P 1·44 0·69
Delvotest MCS L =
18·43 + 10·19C
0·04ST
0·04ST × P 1·04 0·15
Eclipse 100 L =
40·47 + 14·26C
0·04ST
0·02ST × P 3·18 0·36
AMP BRT MRL L =
16·81 + 13·24C
0·06ST
2·26P
0·20ST × P 1·70 0·63
Delvotest MCS L =
4·93 + 2·79C
0·01ST
0·01ST × P 4·47 0·19
Eclipse 100 L =
32·48 + 17·55C
0·07ST × P 4·00 0·15
PEN BRT MRL L =
21·29 + 19·94C
0·05ST
0·05ST × P 2·09 0·55
Delvotest MCS L =
7·53 + 10·04C
0·13ST
0·08ST × P 4·15 0·24
Eclipse 100 L =
11·66 + 11·56C
0·12ST
0·07ST × P 1·11 0·76
OXY BRT MRL L =
96·51 + 0·28C
0·27ST
3·01P
0·14ST × P 0·35 0·95
Delvotest MCS L =
35·26 + 0·15C
0·04ST
0·02ST × P 0·69 0·87
Eclipse 100 L =
29·67 + 0·52C
0·02ST
0·01ST × P 4·09 0·25
AMO: amoxicillin; AMP: ampicillin; PEN: penicillin G; OXY: oxytetracycline; L: ln (Probability (+)/1-Probability (+); C: concentration; β0, β1, β2, β3, β23:
coefficients; ST: storage time (0, 24, 48 or 72 h); P: preservative (no preservative = 1 and azidiol = 0)

when azidiol has been added to milk samples with Eclipse 100: 2 μg/kg), penicillin G (BRT MRL: 1·2 μg/kg;
ampicillin and oxytetracycline, when compared with pre- Delvotest MCS: 1 μg/kg; Eclipse 100: 1 μg/kg) are below
servative-free milk samples. the MRL. The DL for oxytetracycline in the BRT MRL and
Moreover, the interaction between storage time and Delvotest MCS methods (359 and 253 μg/kg, respectively) is
preservative (ST × P) was significant for all the molecules much higher than the MRL (100 μg/kg), but lower (62 μg/kg)
and methods used. The negative sign of the coefficient (β23) for Eclipse 100.
of the interaction (ST × P) indicates that milk samples stored In recent studies, Le Breton et al. (2007) and Stead et al.
with azidiol behave in a different manner from preservative- (2008) validated the use of the Delvotest SP-NT in cow’s
free samples. The highest positive results with the storage milk, calculating detection limits below or equal to the MRL
time are obtained if milk samples contain azidiol. for penicillin, amoxicillin and ampicillin as well as detection
For all the analyses, the goodness-of-fit test shows that limits above the MRL for oxytetracycline. In case of the BRT
the experimental values are similar to those estimated by the test, Sierra et al. (2009b) calculated detection limits below
logistic model, suggesting the suitable adjustment obtained the MRL for six of the ten β-lactams antibiotics tested in
using this model. goat’s milk; penicillin, amoxicillin and ampicillin. The same
For significant effects (P < 0·05), the dose-response curves authors also calculated the DL for the Eclipse 100 test ob-
are displayed in Fig. 1 (amoxicillin), Fig. 2 (ampicillin), Fig. 3 taining limits below the MRL for penicillin, but above the
(penicillin G) and Fig. 4 (oxytetracycline) for preservative- MRLs for amoxicillin and ampicillin.
free samples and with azidiol. To evaluate the effect of storage time of milk samples
As can be appreciated, the increase in storage time of the spiked with antibiotics on the positive results of the microbial
milk samples produces a lower frequency of positive results inhibitor tests, different concentrations (DL: detection
for all methods and antibiotics employed, if the coefficient β2 limit and MRL: Maximum Residue Limit) and storage times
is negative (Table 3). at 4 ± 1 °C (0, 24, 48 and 72 h) were utilised. Table 4 pres-
Regarding the effect of azidiol, the dose-response curves ents these results for each antibiotic and microbial inhibitor
of milk samples with azidiol (II) for each antibiotic are closer test.
to each other if compared with the curves of the preservative- At an equivalent concentration to the DL for each anti-
free samples (I). In the case of milk samples with azidiol and biotic and method, it can be seen that the longer the storage
ampicillin analysed with the Eclipse 100, the refrigeration time (24, 48 and 72 h) at 4 ± 1 °C, the lower the positive
time did not cause any effect, and thus only a dose-response results become for preservative-free milk samples and for
curve can be appreciated in Fig. 2c. This indicates the better those stored with azidiol. But these results remain more
conservation of the antibiotic molecules in those milk constant in all cases if samples are refrigerated with azidiol.
samples stored with azidiol, except for penicillin G, This demonstrates that it is advisable to use this preservative
analysed by the Delvotest MCS and Eclipse 100 methods when the analysis of inhibitors by means of microbial
(Fig. 4b, c). inhibitor tests cannot be carried out on the same day as
The detection limits (DL) for amoxicillin (BRT MRL: collection takes place.
3 μg/kg; Delvotest MCS: 2 μg/kg; Eclipse 100: 3 μg/kg), Moreover, it is worth emphasising that the positive results
ampicillin (BRT MRL: 1·5 μg/kg; Delvotest MCS: 3 μg/kg; in the samples refrigerated with amoxicillin and ampicillin,
 Milk storage and preservation, the response of microbial inhibitor tests 479

a. BRT MRL
I. No preservative II. Preservative
100 100

Positives frequency (%)

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Amoxicillin (µg Kg-1) Amoxicillin (µg Kg-1)

b. Delvotest MCS
I. No preservative II. Preservative
100 100
Positives frequency (%)

80 Positives frequency (%) 80

60 60

40 40

20 20

0 0
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Amoxicillin (µg Kg-1) Amoxicillin (µg Kg-1)

c. Eclipse 100
I. No preservative II. Preservative
100 100
Positives frequency (%)

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Amoxicillin (µg Kg-1) Amoxicillin (µg Kg-1)

Fig. 1. Effect of storage time and azidiol as preservative of the milk samples on the dose-response curves of amoxicillin using microbial
inhibitor tests (Storage time: ◆ 0 h, ■ 24 h, ▲ 48 h and × 72 h). (a) BRT MRL. (b) Delvotest MCS. (c) Eclipse 100.

obtained by analysis with the Delvotest MCS and Eclipse positive results than BRT MRL (0% preservative-free and 5%
100 methods, are more stable than when analysed by BRT with azidiol) when the milk samples were stored for 24 h.
MRL. Conversely, BRT MRL presents a larger number of After 48 h however, the percentage of the positive results
positive results for penicillin G if compared with the other lowers substantially in the Delvotest MCS and Eclipse 100, in
two methods. the case of preservative-free milk samples.
Similarly, in the case of oxytetracycline, the Delvotest Of all the tested penicillins, ampicillin is the molecule in
MCS and Eclipse 100 methods present a better stability of the which positive results were maintained during storage time.
480 M Borràs and others

a. BRT MRL
I. No preservative II. Preservative
100 100

Positives frequency (%)

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 1 2 3 4 5 0 1 2 3 4 5
Ampicillin (µg Kg-1) Ampicillin (µg Kg-1)

b. Delvotest MCS
I. No preservative II. Preservative

100 100

Positives frequency (%)

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 1 2 3 4 5 0 1 2 3 4 5
Ampicillin (µg Kg-1) Ampicillin (µg Kg-1)

c. Eclipse 100
I. No preservative II. Preservative
100 100
Positives frequency (%)
Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 1 2 3 4 5 0 1 2 3 4 5
Ampicillin (µg Kg-1) Ampicillin (µg Kg-1)

Fig. 2. Effect of storage time and azidiol as preservative of the milk samples on the dose-response curves of ampicillin using microbial
inhibitor tests (Storage time: ◆0 h, ■ 24 h, ▲ 48 h and × 72 h). (a) BRT MRL. (b) Delvotest MCS. (c) Eclipse 100.

For penicillin G however, the positive results lowered presents. Roca et al. (unpublished data) employed the
considerably for all three methods when a 48 h storage MS-MS HPLC technique and obtained degradation percent-
time was used. ages for amoxicillin (12·2, 18·2 and 23·9%), ampicillin (6·1,
This decrease in the positive results may be due to 10·4 and 14·5%) and penicillin G (14·4, 18·0 and 21·4%)
degradation or loss of activity in the antibiotic molecules when milk samples were stored at 4 °C for 24, 48 and 72 h,
when storing the milk samples. This fact has been observed respectively. These authors calculated lower degradation
by some authors with techniques that permit the quan- percentages for ampicillin, but higher for penicillin G,
tification of the antibiotic concentration that milk indicating that this antibiotic is more unstable when storage
 Milk storage and preservation, the response of microbial inhibitor tests 481

a. BRT MRL
I. No preservative II. Preservative
100 100

Positives frequency (%)

Positives frequency (%)
80 80

60 60

40 40

20 20

0 0
0 1 2 3 4 0 1 2 3 4
Penicillin G (µg Kg-1) Penicillin G (µg Kg-1)

b. Delvotest MCS
I. No preservative II. Preservative
100 100

Positives frequency (%)

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 1 2 3 4 0 1 2 3 4
Penicillin G (µg Kg-1) Penicillin G (µg Kg-1)

c. Eclipse 100
I. No preservative II. Preservative
100 100
Positives frequency (%)

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 1 2 3 4 0 1 2 3 4
Penicillin G (µg Kg-1) Penicillin G (µg Kg-1)

Fig. 3. Effect of storage time and azidiol as preservative of the milk samples on the dose-response curves of penicillin G using microbial
inhibitor tests (Storage time: ◆ 0 h, ■ 24 h, ▲ 48 h and ×72 h). (a) BRT MRL. (b) Delvotest MCS. (c) Eclipse 100.

takes place at 4 °C. Instability for penicillin G was In the case of oxytetracycline, some authors have
also indicated by Haagsma (1993), who reported con- calculated degradation percentages of between 6–18%
centration losses of over 60% when samples were refriger- (Himanish et al. 2008) and 3·3–10·4% (Roca et al. 2008)
ated for 48 h at 2 °C. This degradation in concentration may at 24 and 48 h of refrigeration with the UV-HPLC and
give rise to a lower percentage of positive results if the DAD-HPLC techniques, respectively. This lower degra-
microbial inhibitor tests in penicillin G are employed, since a dation in the oxytetracycline concentration is reflected
greater instability of this antibiotic is observed when stored in the responses of the Delvotest MCS and Eclipse 100
at 4 °C. methods which, at an equivalent concentration to the DL,
482 M Borràs and others

a. BRT MRL
I. No preservative II. Preservative
100 100

Positives frequency (%)

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Oxytetracycline (µg Kg-1) Oxytetracycline (µg Kg-1)

b. Delvotest MCS
I. No preservative II. Preservative
Positives frequency (%)

100 100

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Oxytetracycline (µg Kg-1) Oxytetracycline (µg Kg-1)

c. Eclipse 100
I. No preservative II. Preservative
100 100
Positives frequency (%)

Positives frequency (%)

80 80

60 60

40 40

20 20

0 0
0 25 50 75 100 125 150 0 25 50 75 100 125 150
Oxytetracycline (µg Kg-1) Oxytetracycline (µg Kg-1)

Fig. 4. Effect of storage time and azidiol as preservative of the milk samples on the dose-response curves of oxytetracycline using microbial
inhibitor tests (Storage time: ◆ 0 h, ■ 24 h, ▲ 48 h and ×72 h). (a) BRT MRL. (b) Delvotest MCS. (c) Eclipse 100.

give positive results at 24 h refrigeration, which at 85 and microbial inhibitor tests will still be able to detect these
91% are still rather high. substances for up to 72 h when stored at 4 °C.
Concerning MRL in general, in the β-lactams tested at an Employing the BRT MRL and Delvotest MCS methods
equivalent concentration to the MRL, positive results did not for oxytetracycline, calculated detection limits (359 and
decrease when milk sample storage time was prolonged 253 μg/kg, respectively) are above MRL (100 μg/kg). Thus,
(Table 4), except for the BRT MRL method for milk samples no positive results were obtained (Table 4) when analysing
with amoxicillin stored preservative-free. Therefore, if milk concentrations equal to MRL. For Eclipse 100, the DL is
samples contain a concentration equivalent to the MRL, lower than the MRL, therefore positive outcomes are
 Milk storage and preservation, the response of microbial inhibitor tests 483

Table 4. Effect of storage time and preservative on the positive results (%) of microbial inhibitor tests in milk samples for different antibiotics
BRT MRL DELVOTEST MCS ECLIPSE 100

DL MRL DL MRL DL MRL

Antibiotics (MRL) Storage time at 4 °C (h) NP P NP P NP P NP P NP P NP P

AMO (4 μg/kg) 0 95 95 100 100 95 95 100 100 95 95 100 100
24 51 88 98 100 77 89 100 100 82 88 100 100
48 6 77 77 99 37 78 100 100 51 74 100 100
72 0 60 15 99 9 60 100 100 19 52 100 100
AMP (4 μg/kg) 0 95 95 100 100 95 95 100 100 95 95 100 100
24 30 85 100 100 92 94 100 100 73 93 100 100
48 0 59 100 100 86 92 99 100 36 93 100 100
72 0 28 100 100 76 90 99 99 10 93 100 100
PEN (4 μg/kg) 0 95 95 100 100 95 95 100 100 95 95 100 100
24 53 79 100 100 18 61 100 100 20 62 100 100
48 8 50 100 100 0 7 100 100 0 9 100 100
72 0 22 100 100 0 0 100 100 0 0 100 100
OXY (100 μg/kg) 0 95 95 0 0 95 95 0 0 95 95 100 100
24 0 5 0 0 77 85 0 0 77 91 100 100
48 0 0 0 0 42 68 0 0 39 85 100 100
72 0 0 0 0 13 42 0 0 10 76 100 100
AMO: amoxicillin; AMP: ampicillin; PEN: penicillin G; OXY; oxytetracycline; NP: no preservative; P: preservative (azidiol); DL: detection limit; MRL: Maximum
Residue Limit

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Journal of Dairy Research (2013) 80 484. © Proprietors of Journal of Dairy Research 2013
doi:10.1017/S0022029913000563

ERRATUM

Effect of storage and preservation of milk samples on the response of

microbial inhibitor tests – ERRATUM
Milagro Borràs Llopis, Marta Roca Marugán, Rafael Lisandro Althaus and Maria Pilar Molina Pons

doi: 10.1017/S0022029913000423 Published Cambridge University Press, 9 October 2013

The name of the second author is Marta Roca Marugán and not Marugón as published.

Reference
Borràs. et al. (2013) Effect of storage and preservation of milk samples on the response of microbial inhibitor tests. Journal of Dairy Research 80 475–484