Effect of storage and preservation of milk samples on the response of microbial inhibitor tests Milagro Borràs Llopis 1 *, Marta Roca Marugón 2 , Rafael Lisandro Althaus 3 and Maria Pilar Molina Pons 1 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 have public health implications, such as the development of allergic reactions, interference in the intestinal flora and the development of antibiotic resistance (Alanis, 2005; Demoly & Romano, 2005). Moreover, it can interfere with the manu- facture of dairy products (Babu et al. 1989; Berruga et al. 2007). To prevent these problems, the European Union (Regulation, 853/2004/EC) specified that raw milk intended for human consumption must not contain antimicrobial resi- dues exceeding the Maximum Residue Limits (MRLs) as established by Regulation (2010/37/EC). Different microbial inhibitor tests have been developed for the detection of these residues in milk. The principle of these methods is based on the inhibition of the growth of the Geobacillus stearothermophilus subsp. calidolactis spore. This inhibition of growth is demonstrable as the colour of the culture medium remains unchanged. In inhibitor-free milk, the test microorganism grows and a colour change in the indicator takes place. These tests are widely utilised for the screening of inhibitors in milk, as they are quick, easy to use and economical (Toldrá & Reig, 2006; IDF, 2010). In addition, they have the sensitivity to detect β-lactams and sulphonamides, but they are not sensitive enough to detect many other antibiotics such as tetracyclines, quinolones, macrolides and aminoglycosides (Le Breton et al. 2007; Kantiani et al. 2009; Navrátilova, 2009; Perme et al. 2010). Moreover, accuracy in milk inhibitor analysis is very im- portant to most dairy farmers and to the dairy industry. Many factors can affect the response of these tests, such as milk composition, somatic cell count and total bacteria count (Althaus et al. 2003; Kang et al. 2005; Comunian et al. 2010). Also, other important factors can affect the results namely adequate milk sampling as well as storage before analysis in control laboratories. 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 state of the sample at the time of sampling is not adversely *For correspondence; e-mail: miborllo@upvnet.upv.es Journal of Dairy Research (2013) 80 475–484. © Proprietors of Journal of Dairy Research 2013 doi:10.1017/S0022029913000423 475