1) The need to verify the kinetics of the response and the presence of a single effector before deciding that we are looking at a case of hormesis. In a previous work [21], we demonstrate that the response is a sigmoidal function of time for the same reasons for which it is a sigmoidal

function of dose (the most sensitive elements of the population not only respond at lower doses but also at shorter times). Therefore, the examination of the time-course of the response, in any case with a well A-1210477 defined toxicological interest, is especially important if anomalies are detected in an assay at only one exposure time. 2) The inadequacy of the plate assays based on inhibition zones. These are qualitatively useful, but too imprecise to detect the effects mentioned here. 3) The need to confirm carefully the antimicrobial

effects of the bacteriocins in the specific conditions of their application, when they are used as agents for the control of undesirable microbiota in food products. Methods Reagents The tested agents were nisin, phenol (both from SIGMA) and pediocin. The last was prepared from a Pediococcus acidilactici NRRL B-5627 culture in MRS medium, according to the process described by Vázquez et al. [22]. Microorganisms and bioassay The microorganisms used were Carnobacterium piscicola CECT 4020 and Leuconostoc mesenteroides subsp. lysis (kindly provided by Dr. Ray, University of Wyoming, Laramie, USA), both MCC950 nmr commonly Inositol monophosphatase 1 used as indicators in bacteriocin bioassays. Experiments were carried out in quadruplicate, using methods which were described in detail in previous studies [23–25]. To prepare the C188-9 price microbial suspensions, cultures aged 12 h in MRS medium were centrifuged, the sediment washed with 0.05 M, pH = 6.0 biphtalate-NaOH buffer in fresh MRS medium (MRS-f), and the washed sediment resuspended in

MRS-f and adjusted to an absorbance (700 nm) of 0.200. For DR analysis, four series of dilutions in MRS-f were prepared with each effector, and the assay began combining equal volumes (1 ml) of microbial suspension and effector solution (MRS-f in the control). Incubations were performed in 15 ml tubes at 23, 30 and 37°C, with 200 rpm orbital shaking, and the results were quantified as R = 1-(A D/A 0), where A 0 and A D are the absorbances at 700 nm of the control and the dose D respectively. The inhibitory and stimulatory responses have thus positive and negative sign, respectively. For comparative purposes, A D and A 0 quantifications were performed in some cases by plate count on MRS-agar with similar results to those obtained from absorbances (data not shown). However, attempts to carry out systematic inhibition bioassays by means of the usual plate method of the clear zones (halos) produced qualitatively similar, but more inaccurate results.