Sterile horse blood

S. downii sp. nov. strain CECT 9732T, isolated from a supragingival dental biofilm sample of an individual with Down syndrome [29 (link)], and the reference bacterial strains S. mutans ATCC 25175, V. parvula NCTC 11810, Actinomyces naeslundii ATCC 19039, F. nucleatum DMSZ 20482, A. actinomycetemcomitans DSMZ 8324, and P. gingivalis ATCC 33277 were used to develop a multi-species biofilm model. Bacteria were cultured anaerobically (10% H₂, 10% CO₂, and balance N₂) on blood agar plates (Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid, Basingstoke, UK), 5.0 mg mL⁻¹ hemin (Sigma-Aldrich, St. Louis, MO, USA) and 1.0 mg mL^-1 menadione (Merck, Darmstadt, Germany) for 72 h at 37 °C.

The Enterococcus faecalis (CCARM 5511), Enterococcus faecium (KACC 11954), Porphyromonas gingivalis (KCTC 5352), Streptococcus mutans (KACC16833), Streptococcus sobrinus (KCTC5809), Staphylococcus aureus (CCARM3506), Staphylococcus epidermidis (KACC 13234), Fusobacterium nucleatum (KCTC 2640) and Actinomyces viscosus (KCTC 9146) strains used in this study are listed in Table 1. E. faecalis, E. faecium, S. epidermidis, S. aureus and A. viscosus were maintained in tryptic soy broth (TSB). S. mutans and S. sobrinus were maintained in brain heart infusion broth (BHI). P. gingivalis was maintained in tryptic soy broth supplemented with 10% defibrinated horse blood. F. nucleatum was maintained in reinforced clostridial agar and incubated at 37 °C. A. viscosus, S. sobrinus, F. nucleatum and P. gingivalis bacteria were cultured in brain heart infusion agar plate (BHI), reinforced clostridial agar plate, tryptic soy agar plated (TSB) and blood agar plates (Blood Agar Oxoid No₂; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid) at 37 °C for 24–72 h in anaerobic conditions (10% H₂, 10% CO₂, and balanced N₂) [21 (link),22 (link),23 (link)].

Methodology for developing the multispecies biofilm was similar to that previously reported from our research group [14 (link), 23 (link)]. Briefly, reference strains of P. gingivalis ATCC 33277, Streptococcus oralis CECT 907T, Actinomyces naeslundii ATCC 19039, Veillonella parvula NCTC 11810, Fusobacterium nucleatum DMSZ 20482 and Aggregatibacter actinomycetemcomitans DSMZ 8324 were used. Each bacterial strain was grown on blood agar plates (blood agar Oxoid no. 2; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid), 5.0 mg/L hemin (Sigma, St Louis, MO, USA) and 1.0 mg/L menadione (Merck, Darmstadt, Germany) under anaerobic conditions (10% H₂, 10% CO₂ and 80% N₂) at 37°C for 24–72 h.