Schaedler agar plates

Twenty P. gingivalis strains from the strain collection of the Laboratory of Oral Microbiology (University of Bern, Department of Periodontology) were included in the screening experiments determining minimal inhibitory concentration (MIC) values of the honeys. Strains included were two laboratory strains (ATCC 33277, W83) and 18 banked as isolates from periodontitis samples (BGH40-2, D2-4-3, D5-2-2, J358-1, J361-1, J362-1, J374-1, J378-1, J424-1, J426-1, J430-1, J435-1, J439-1, M5-1-2, MaRL, PL55, PL110, PL126). In the follow-up experiments the type strain ATCC 33277 and three other strains (M5-1-2, MaRL, J361-1) were used. The selection was based on the different colony morphology. M5-1-2 strain forms smooth colonies, MaRL very rough ones and J361 colonies similar to those formed by the type strain. The identity was confirmed by 16S rDNA sequence analysis.
All strains were kept frozen prior to the experiments. They were transferred and cultured anaerobically (10% H₂, 5% CO₂, 85% N₂) on Schaedler agar plates (Oxoid, Basingstoke, GB) containing 8% sheep blood 24 h before starting the experiments at 37°C.

The dentin discs on the plastic specimens were colonized with a biofilm consisting of 12 bacterial strains (Streptococcus gordonii ATCC 10,558, Actinomyces naeslundii ATCC 12,104, Fusobacterium nucleatum ATCC 25,586, Campylobacter rectus ATCC 33,238, Eubacterium nodatum ATCC 33,099, Eikenella corrodens ATCC 23,834, Parvimonas micra ATCC 33,270, Filifactor alocis ATCC 33,099, Prevotella intermedia ATCC 25,611, Porphyromonas gingivalis ATCC 33,277, Tannerella forsythia ATCC 43,037, Treponema denticola ATCC 35,405). Bacterial strains (except for T. denticola) were cultivated on Schaedler Agar plates (Oxoid Basingstoke, UK) with 5% sheep blood in an anaerobic atmosphere or with 5% CO₂. T. denticola was maintained in mycoplasma broth (BD, Franklin Lakes, NJ) supplemented with niacinamide, spermine tetrahydrochloride, and cocarboxylase in anaerobic conditions.
Dentin specimens were covered with 1.5% bovine serum albumin for 15 min, before they were placed into tubes with nutrient broth (Wilkins-Chalgren broth with nicotinamide adenine dinucleotide and N-acetyl muramic acid) and bacteria. The tubes have been incubated in anaerobic conditions at 37 °C for 3.5 days. After 48 h, P. gingivalis ATCC 33,277, T. forsythia ATCC 43,037, and T. denticola ATCC 35,405 were added again to guarantee a colonization of these bacteria in biofilm.

A 12-species periodontal biofilm was used in this study:

Streptococcus gordonii ATCC 10558

Actinomyces naeslundii ATCC 12104

Fusobacterium nucleatum ATCC 25586

Campylobacter rectus ATCC 33238

Parvimonas micra ATCC 33270

Eikenella corrodens ATCC 23834

Treponema denticola ATCC 35405

Prevotella intermedia ATCC 25611

Capnocytophaga gingivalis ATCC 33624

Porphyromonas gingivalis ATCC 33277

Tannerella forsythia ATCC 43037

Filifactor alocis ATCC 33099

All strains (except for T. denticola which was maintained in Mycoplasma broth (BD, Franklin Lake, NJ)) were cultured on Schaedler agar plates (Oxoid, Basingstoke, UK) with 5% sheep blood, in an anaerobic incubator or with 5% CO₂ (S. gordonii) at 37 °C. The bacteria were suspended in 0.9% w/v NaCl according to McFarland 4. One part S. gordonii was mixed with two parts A. naeslundii, and four parts of the other nine species.