The fifteen multi-species biofilm that were used to contaminate the titanium discs are the essential bacterial strains found in peri-implantitis sites. The included bacterial strains were Porphyromonas gingivalis (ATCC 33277), Aggregatibacter actinomycetemcomitans (ATCC43718), Prevotella intermedia (ATCC 25611), Fusobacterium nucleatum (ATCC10953), Streptococcus mutans (ATCC 25175), and Streptococcus sobrinus (ATCC 33478). Commensal bacteria were also included in the study in order to mimic as possible the peri-implant biofilm. The commensal bacteria were Actinomyces viscosus (ATCC 15987), Streptococcus cristatus (ATCC 49999), Actinomyces naeslundii (ATCC 51655), Streptococcus mitis (ATCC 49456), Streptococcus oralis (DSM 20627), Streptococcus sanguinis (LMG 14657), Streptococcus gordonii (ATCC 49818), Veillonella parvula (DSM 2008), and Streptococcus parasanguinis (DSM 6778).
Streptococcus sobrinus
Manufactured by American Type Culture Collection
Streptococcus sobrinus is a bacterial strain used in research and laboratory applications. It is a Gram-positive, coccoid-shaped bacterium that belongs to the Streptococcus genus. This strain is commonly used for studying the biology, genetics, and pathogenicity of oral bacteria.
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Lab products found in correlation
Streptococcus mutans, by American Type Culture Collection (4 mentions)
Streptococcus mitis, by American Type Culture Collection (3 mentions)
Streptococcus sanguinis, by American Type Culture Collection (3 mentions)
Streptococcus salivarius, by American Type Culture Collection (2 mentions)
Streptococcus gordonii, by American Type Culture Collection (1 mentions)
Actinomyces viscosus, by American Type Culture Collection (1 mentions)
Bacillus subtilis, by American Type Culture Collection (1 mentions)
Pseudomonas aeruginosa, by American Type Culture Collection (1 mentions)
Bacteroides fragilis, by American Type Culture Collection (1 mentions)
Prevotella nigrescens, by American Type Culture Collection (1 mentions)
Actinomyces naeslundii, by American Type Culture Collection (1 mentions)
Chlorhexidine digluconate, by Merck Group (1 mentions)
Resazurin, by Merck Group (1 mentions)
4 protocols using streptococcus sobrinus
1
Multi-species Biofilm Peri-implantitis Model
2
Antibacterial Metabolites Profiling
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The inhibitory metabolites produced by the bacteria tested were inferred and detected using a well-diffusion technique10 (link). The chosen strains were grown in overnight cultures on MRS agar for 24 h at 37 °C. The study's indicator microorganisms, which were obtained from the Persian Type Culture Collection (PTCC), were Streptococcus sanguinis PTCC 1449, Streptococcus salivarius PTCC 1448, Streptococcus sobrinus PTCC 1601, Yersinia enterocolitica ATCC 23715, Pseudomonas aeruginosa PTCC 1181, Staphylococcus aureus ATCC 25923, Streptococcus mutans PTCC 1683, Bacillus subtilis ATCC 19652, Klebsiella pneumoniae PTCC 1053, Listeria monocytogenes ATCC 13932, Shigella flexneri PTCC 1234, and Escherichia coli PTCC 1276. Half a McFarland of indicator bacteria (1.5 108 CFU mL−1) was poured into various appropriate media, and wells were cut into plates. The plates were then incubated overnight at 37 °C with each well filled with 50 µL filtered supernatant, and a digital caliper was used to estimate the inhibitory zone around the wells.
3
Antibacterial Evaluation of Essential Oils
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The tested strains were obtained from the American Type Culture Collection (ATCC, Rockville MD, USA). The following microorganisms were used in the evaluation of the antibacterial activity of the essential oils: Streptococcus mitis (ATCC 49456), Streptococcus mutans (ATCC 25175), Streptococcus sanguinis (ATCC 10556), Streptococcus sobrinus (ATCC 33478), Actinomyces naeslundii (ATCC 19039), Bacteroides fragilis (ATCC 25285), and Prevotella nigrescens (ATCC 33563).
4
Antimicrobial Activity of Essential Oils
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Bacteria were acquired from the American Type Culture Collection (ATCC) and kept in the culture collection of the Laboratory of Research on Applied Microbiology (LaPeMA) at Universidade de Franca, state of São Paulo, Brazil, at -80°C. The following microorganisms were used: Streptococcus salivarius (ATCC 25975), Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49452), Streptococcus sanguinis (ATCC 10556) and Streptococcus sobrinus (ATCC 33478).
Minimum Inhibitory Concentrations (MIC) of essential oils were determined by the broth microdilution method in 96-well microplates, following the methodology of CLSI (2006) . Samples were dissolved in 125 µL tryptic soy broth (TSB) to yield compound concentrations between 50 and 400 µg/mL. The inoculum was adjusted to 625 nm for every microorganism in a spectrophotometer to obtain cell concentration of 5 × 10 5 colony-forming units (CFU/mL) (CLSI 2006) . Chlorhexidine digluconate (Sigma-Aldrich), at concentrations from 0.115 to 59.0 µg/mL, was used as the positive control. The microplates were incubated at 37 °C for 24 h; then, 30 µL 0.02% resazurin (Sigma-Aldrich) aqueous solution was added to every well (Sarker et al., 2007) . resazurin is an oxireduction probe that enables microbial growth to be immediately observed. Blue and red colors represent the absence and the presence of microbial growth, respectively.
Minimum Inhibitory Concentrations (MIC) of essential oils were determined by the broth microdilution method in 96-well microplates, following the methodology of CLSI (2006) . Samples were dissolved in 125 µL tryptic soy broth (TSB) to yield compound concentrations between 50 and 400 µg/mL. The inoculum was adjusted to 625 nm for every microorganism in a spectrophotometer to obtain cell concentration of 5 × 10 5 colony-forming units (CFU/mL) (CLSI 2006) . Chlorhexidine digluconate (Sigma-Aldrich), at concentrations from 0.115 to 59.0 µg/mL, was used as the positive control. The microplates were incubated at 37 °C for 24 h; then, 30 µL 0.02% resazurin (Sigma-Aldrich) aqueous solution was added to every well (Sarker et al., 2007) . resazurin is an oxireduction probe that enables microbial growth to be immediately observed. Blue and red colors represent the absence and the presence of microbial growth, respectively.
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