Culturing Diverse Bacterial Species

Lactobacillus reuteri DSM 17938 [37 (link)] kindly provided by BioGaia AB (Stockholm, Sweden), was used in the study. The strain was plated on DeMan, Rogosa, and Sharpe agar (MRSA; Oxoid Limited, Basingstoke, Hampshire, UK), and incubated at 37 °C for 24 h in an anaerobic atmosphere (Anaerogen Pak Jar, Oxoid Ltd.). Different media and growth conditions were selected for the different bacterial species used in the study. In particular, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were cultured on Mueller-Hinton agar (MHA; Oxoid Ltd.) for 24 h in aerobic conditions; Fusobacterium nucleatum was cultured on Fastidious Anaerobe Agar (FAA; Lab M, Heywood, UK) and 5% (v/v) of defibrinated horse sterile blood (Oxoid Ltd.) for 48 h in anaerobiosis (Anaerogen Pak Jar, Oxoid Ltd.); and Streptococcus mutans was cultured on Columbia agar (CA; Oxoid Ltd.) and 5% (v/v) of defibrinated horse sterile blood (Oxoid Ltd.) for 24 h at 5% CO₂. The isolated strains have been previously used in other research studies [33 (link)]. The study did not require ethical approval because all isolates were obtained as a part of routine diagnostic microbiology investigations, however, patients gave informed consent for further scientific studies.