Polaron e5000

Untreated KPC cells (negative control), those treated with imipenem (positive control), and those treated with ZnO-NPs (tested sample) were characterized by SEM to study the morphological cell alterations. The concentration of imipenem for positive control was 500 mg/ml, while for the tested bacteria, the MIC concentration was 0.5 mg/ml. The treated KPC cells were fixed with 2.5% glutaraldehyde in a phosphate buffer with pH 7.2. The samples were post-fixed in 1% osmium tetroxide, followed by dehydration through an ascending ethanol series to critical point drying and coating with Au–Pd (80:20) using a Polaron E5000 sputter coater (Quorum Technologies, Laughton, UK). The samples were analyzed by SEM (FEI Quanta 250) at an accelerating voltage of 25 kV using an SE detector (Kim et al., 2014 (link); Kelly et al., 2017 (link)).

Samples of A. salina nauplii—employed in the lethality test—and samples of rat erythrocytes—obtained from the hemolysis assay—were examined for morphological alterations induced by the aqueous extract of M. alcicornis. Some samples were dyed with starch blue and observed directly through light microscopy. For observations by SEM and TEM, samples were fixed in filtered sea water or isotonic saline solution containing 3 % glutaraldehyde and 0.1 M of sodium cacodylate, postfixed in 2 % OsO₄ in cacodylate buffer, and dehydrated in an ethanol series. Then, for TEM analysis, samples were embedded in Epon epoxy resin and the blocks obtained were sliced (60 nm) in an ultramicrotome (Mtx RMC Boeckler Instruments, USA) and contrasted with uranyl acetate and lead citrate. These sections were observed in an electron microscope (JEM 1010, JEOL, USA) operated at 80 kV. For SEM analysis, the samples were dried in a critical-point dryer (Polaron E5000, Quorum Technologies, UK), covered with carbon in an evaporator (JEE4X, JEOL, USA) and with a thin sheet of gold in an ion sputterer (Polaron 11-HD, Quorum Technologies, UK), and finally observed with a scanning electron microscope (DMS 950, Zeiss International) at an accelerating voltage of 20 to 25 kV.