Standard reference strain P. gingivalis ATCC 33277 was selected for the present study. Bacteria were 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) in anaerobic conditions (10% H2, 10% CO2, and balance N2) at 37°C for 72 hours.
Blood agar oxoid no 2
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Blood Agar Oxoid No 2 is a general-purpose culture medium used for the isolation and identification of a variety of microorganisms, including both aerobic and anaerobic bacteria. It contains defibrinated blood, which supports the growth of fastidious organisms and allows for the detection of hemolytic reactions.
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9 protocols using blood agar oxoid no 2
1
Cultivation of Porphyromonas gingivalis
2
Cultivation of Oral Bacterial Strains
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Six bacterial strains, including Streptococcus oralis CECT 907 T, Veillonella parvula NCTC 11810, Actinomyces naeslundii ATCC 19039, F. nucleatum DMSZ 20482, A. actinomycetemcomitans DSMZ 8324 and P. gingivalis ATCC 33277 were used. Bacteria were cultured in blood agar plates (Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid), 5.0 mg L− 1 hemin (Sigma, St. Louis, MO, USA) and 1.0 mg L− 1 menadione (Merck, Darmstadt, Germany) at 37 °C for 24–72 h in anaerobic conditions (10% H2, 10% CO2, and balance N2).
3
Multi-species Biofilm Model Development
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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.
4
Culturing Oral Anaerobes on Blood Agar
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S. oralis CECT 907T, V. parvula NCTC 11810, A. naeslundii ATCC 19039, F. nucleatum DMSZ 20482, P. gingivalis ATCC 33277 and A. actinomycetemcomitans DSMZ 8324 were 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 haemin (Sigma, St. Louis, MO, USA) and 1.0 mg/L menadione (Merck, Darmstadt, Germany) at 37 °C for 48 h under anaerobic conditions (10% H2, 10% CO2 and N2 balance).
5
Growth conditions for oral anaerobes
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Reference strains of S. oralis CECT 907T, V. parvula NCTC 11810, A. naeslundii ATCC 19039, F. nucleatum DMSZ 20482, A. actinomycetemcomitans DSMZ 8324, and P. gingivalis ATCC 33277 were used. These bacteria were 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−1 hemin (Sigma, St. Louis, MO, USA) and 1.0 mg L−1 menadione (Merck, Darmstadt, Germany) in anaerobic conditions (10% H2, 10% CO2, and balance N2) at 37 °C for 24–72 h.
6
Cultivation of Oral Anaerobes
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Reference strains of Streptococcus oralis CECT 907T, Veillonella parvula NCTC 11810, Actinomyces naeslundii ATCC 19039, F. nucleatum DMSZ 20482, Aggregatibacter actinomycetemcomitans DSMZ 8324, and P. gingivalis ATCC 33277 were used. These bacteria were 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−1 hemin (Sigma, St. Louis, MO, USA) and 1.0 mg L−1 menadione (Merck, Darmstadt, Germany) in anaerobic conditions (10% H2, 10% CO2, and balance N2) at 37 °C for 24–72 h.
7
Cultivation and Maintenance of Oral Bacteria
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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)].
8
Multispecies Biofilm Development Protocol
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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.
9
Culturing Porphyromonas gingivalis
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Porphyromonas gingivalis ATCC 33277 was used in this study and grown on supplemented blood agar plates [Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK; with 5.0 mg/L hemin (Sigma, St. Louis, MO, USA), 1.0 mg/L menadione (Merck, Darmstadt, Germany) and 5% (v/v) sterile horse blood (Oxoid)], at 37 °C for 48 h in anaerobiosis (10% H2, 10% CO2, and 80% N2).
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