Physiological saline solution

Staphylococcus aureus (ATCC 25923, purchased from Spanish Type Culture Collection) was cultured in Petri dishes (90 × 15 mm) containing Mueller–Hinton medium (bioMérieux, Spain) and incubated for 24 h at 37 °C. Several colonies (8–12 depending on size) were collected and transferred onto 5 mL of different media (Tryptic Soy Broth (TSB, bioMérieux, Spain)) or physiological saline solution (0.9% NaCl in water, Sigma-Aldrich, Spain). To standardize the number of bacteria in the suspension, we used a standard McFarland suspension of 4, which equals approximately 1.2 × 10⁹ Colony-Forming Units (CFU) per millilitre (or OD = 1 at 540 nm). From this initial suspension, decimal dilutions were made for use in the corresponding experiment (10⁸, 10⁷ and 10⁶ CFU/mL) (Fig. 1).Figure 1

Flow-chart of the experimental design.

The Staphylococcus aureus strain ATCC 25923, purchased from the Spanish Type Culture Collection, was used in the present study. The microorganism was cultured in Petri dishes (90 × 15 mm) containing Mueller–Hinton medium (bioMérieux, Spain), and incubated for 24 h at 37 °C. For each experimental setup, several colonies (8 to 12 depending on size) were collected and transferred onto 5 mL of sterile physiological saline solution (0.9% NaCl in water, Sigma-Aldrich, Spain). To standardize the number of bacteria in the suspension, we used a McFarland suspension of 4, which equals approximately 1.2 × 10⁹ Colony-Forming Units (CFU) per milliliter (or OD = 1 at 540 nm). The volume of bacterial suspension added to tubes is mentioned in Fig. 1 (125 ml).Figure 1

Flow diagram showing an experimental design to determine the bactericidal effect of NE used in the different in vitro experiments.

The study protocol was reviewed and approved by the recommendations of the Animal Care Committee of the University of Debrecen, Hungary, according to national laws and European Union regulations (European Communities Council Directive of 24 November 1986 (86/609/EEC)], and was properly conducted according to the University’s Guidelines for Animal Experimentation. All animals were kept under standard conditions with chow and water ad libitum. The experiments were performed on male Wistar–Kyoto rats (Gödöllő, Hungary). The animals were divided into experimental groups: experimental group 1 (12 control rats) and experimental group 2 (21 CFA-treated animals). In animals in the treated group, chronic inflammation was induced by intra-plantar injection of 100 µL 1:1 mixture of physiological saline solution and CFA (Sigma, St Louis, MO, USA) into the right hind paw, according to the method described earlier [65 (link)].