Brain heart infusion agar

The reference species of following fungi and bacteria were obtained from the American Type Culture Collection (ATCC), the Centraalbureau voor Schimmelcultures (CBS), and Teikyo University Institute of Medical Mycology (TIMM): Aspergillus flavus (ATCC® 64025™), A. fumigatus (ATCC® 14110™), A. clavatus (CBS® 514.65™), A. niger (TIMM® 0113™), Pseudomonas aeruginosa (ATCC® 27853™), and Enterococcus faecalis (ATCC® 11700™). Fungi were used for the biosynthesis of silver nanoparticles and bacteria were used as a positive control in nitrate reduction test.
Fungi were subcultured in Sabouraud Dextrose Agar (Merck, Germany) and incubated at 35°C for 24 hours. In order to prevent the bacterial growth, 50 mg/L chloramphenicol (Sigma-Aldrich, Germany) was added to the cultures. Bacteria species were subcultured in Brain Heart Infusion Agar (Merck, Germany) and were incubated at 37°C for 24 hours.

The formation of the biofilm was assessed qualitatively by culturing enterococcal isolates on CRA plates [24 (link),25 (link)]. To prepare CRA plates, 1 L of brain heart infusion agar (Merck, Darmstadt, Germany) was mixed with 0.8 g of Congo red dye (Merck, Darmstadt, Germany), and 36 g of saccharose. The plates were incubated for 24 h at 37 °C and followed overnight at room temperature. Black colonies were assessed to be strains with a high capacity to produce a biofilm (P; producer), whereas strains with red colonies were identified as the strains incapable of producing the biofilm (NP; non-producer).

Twenty-five grams of meat samples was homogenized for 90 s in a stomacher (Heidolph, Schwabach, Germany) with 225 ml of peptone water (PW) containing 6.5% NaCl and then incubated at 37°C for 24 h. After primary enrichment, a loopful (without shaking the flask) from each of the enriched homogenates was streaked onto Baird-Parker agar (MERCK, Darmstadt, Germany) supplemented with 5% egg yolk and tellurite and incubated under aerobic conditions at 37 °C for 24 h. Colonies with typical gray-black appearance surrounded by a clear zone were enumerated as coagulase-positive staphylococci and sub-cultured onto Mannitol salt agar. Clinical specimens were also cultured onto Brain Heart Infusion agar and Mannitol salt agar (MERCK, Darmstadt, Germany) [22 (link), 23 (link)]. The isolates were identified as S. aureus by further biochemical characterization using Gram stain, catalase, coagulase, oxidase, lipase, DNase, and PCR targeting the S. aureus-specific femA gene (S. aureus species specific).

One g of fresh feces sample was suspended in 99 mL Ringer solution (6.5 g NaCl, 0.42 g KCl, 0.25 g CaCl₂, and 0.2 g of NaHCO₃) supplemented with 0.05% L-cystein HCL, followed by serial 1:10 dilution in Ringer solution. One hundred microliters of appropriate dilutions were spread on different selective media and incubated anaerobically as follows: total anaerobic bacteria [brain heart infusion agar (Merck, Darmstadt, Germany), 37°C/48 h]; total lactobacilli [LAMVAB agar Hartemink et al. (1997 (link)), 37°C/72 h], total Enterobacteriaceae [crystal violet agar (Merck, Darmstadt, Germany), 37°C/24 h; total clostridia (reinforced clostridial agar, Becton and Dickinson, Heidelberg, Germany) 37°C/48 h].