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Briefly, bacterial suspensions were prepared from overnight cultures in BHI. Bacterial concentration was adjusted by measuring optical density at 600 nm to 0.1 for all three bacterial strains. An aliquot from each bacterial suspension was then serially diluted and plated for checking the colony purity and number of colony forming units, CFUs.

A bacterial mix was prepared in a sterile jar by pooling bacterial suspensions of P .

Each well was inoculated with 300 ?l bacterial mix and 1,200 ?l fresh BHI. The amount of bacteria in the inoculum were calculated as: 1.83 ? 10 7 ± 5.79 ? 10 6 CFU/ml F . gingivalis , and 4.43 ? 10 7 ± 1.44 ? 10 7 CFU/ml A . actinomycetemcomitans (mean and standard deviation).

The plates were incubated anaerobically to allow biofilm formation for 72 hr. The biomass of the biofilm and the viability of bacteria were checked at different time points in preliminary tests in order to develop a mature biofilm for exposure to antibiotics.

Bacterial mass in the biofilms was assessed by plate reading by plate reader (Synergy H1 Hybrid reader, BioTek instruments, USA).

The biofilm biomass absorbance was measured at 600 nm immediately after complete growth medium removal.

The viability of bacteria in biofilm was assessed by

culture

CFUs

First, the growth medium and planktonic cells were removed from the 3?day old biofilms, each biofilm was scrapped off the well by use of a sterile cell scraper (Sarstedt, Newton, USA), suspended in 1 ml fresh BHI, and disrupted by vigorous pipetting. The biofilm suspensions were 10?fold diluted on FAA plates in order to obtain plates containing CFUs in range of 1–500 colonies. After 5–7 days of incubation in anaerobic conditions, CFUs were enumerated on FAA plates by help of a stereomicroscope, distinguished by the colony morphology. The biofilm development and structure were assessed by confocal laser scanning microscopy (CLSM). Prior to examining under CLSM, the biofilms grown

for

Fully hydrated biofilms were stained for 20–30 min in the dark, in anaerobic conditions, thereafter immediately examined by CLSM (Leica TCS SP5; Leica Microsystems, Germany) equipped with an oil?immersion of 63? or 100? objective (Carl Zeiss, Jena, Germany) with beam path settings at 488 nm and 543 nm, respectively.

Fluorescence intensity thresholds were manually set for each fluorescent color when examining a negative control biofilm and not further modified. At least three different areas on each biofilm were investigated.

Image stacks were analyzed with the Leica Confocal Lite ® software (Leica Microsystems).

Antibiotic cocktails to be used in assays were freshly prepared in BHI prior to each experiment. The antibiotic concentrations tested in this study were defined as “high concentration” when combinations used were AMX + MET (1,080 ?g/ml + 2,160 ?g/ml) and PV + MET (1,080 ?g/ml + 2,160 ?g/ml); and “low concentration” when AMX + MET (360 ?g/ml + 720 ?g/ml), respectively, PV + MET (360 ?g/ml + 720 ?g/ml) were used.

Growth medium together with planktonic bacteria were carefully removed and antibiotic solutions in BHI at given concentrations were gently

placed

Then, the growth medium containing antibiotics or CHX were removed and each biofilm was harvested in 1 ml fresh BHI and disrupted as described above.

Viable counts were recorded on FAA plates incubated for 5 days. Paired t test was used to investigate significant differences between the two different combinations of antibiotics.

A p value less than .05 was considered statistically significant. All three types of bacterial strains were harvested from 3?day old biofilms. The viable bacteria in biofilms were mainly represented by F .

gingivalis, which showed a nearly two?log increase in numbers during the 3 days incubation.

actinomycetemcomitans declined in the biofilm compared to the initial inoculum per well, as depicted in Figure 1. The bacterial mass of the 3?day old biofilm as measured by plate reading ( n = 7) was 0.568 ± 0.075 AU.

A mature biofilm with three?dimensional structure with mushroom?like shapes and nutrient channels was formed after 3 days incubation in anaerobic conditions. Both live and dead bacterial?like structures could be observed by CLSM as shown in Figures 2 and 4a. Exposure of biofilms to CHX for 2 hr resulted in eradication of all three bacterial strains, as no CFU could be detected at plate counting. A shorter exposure time than 2 hr to CHX 0.2% resulted in incomplete elimination of live bacteria from the biofilms; while exposure for 2 hr to CHX 0.2% diluted in 1:2 or 1:4 volumes resulted also in detection of CFU after harvesting of positive?control biofilms (results not shown). gingivalis were completely eliminated from the biofilms by antibiotic treatment, both AMX + MET and PV + MET in high and low concentrations, as presented in Figure 3.

actinomycetemcomitans was five logs reduced following exposure of biofilms to high concentration of antibiotics and four logs following low concentration. There was no statistical difference (paired t test) between the AMX + MET and PV + MET combinations with respect to growth inhibition of A .

actinomycetemcomitans neither in the high concentration ( p = .95) nor in the low concentration ( p = .17). The combination PV + MET in high concentration had significantly stronger effect in eliminating A .

actinomycetemcomitans than the low concentration ( p = .041). No significance was achieved when comparing between the effect of low and high concentrations of AMX + MET ( p = .076).