Australasian Dentist Magazine March April 2021

Category 44 Australasian Dentist Planktonic assay The two formulations of Citrox ® (BC30 and MDC30) were first assessed with regard to their antimicrobial activity against planktonic suspensions of the test species. In these experiments an overnight culture of each strain was prepared in the appropriate liquid medium to a turbidity level equal to a MacFarland standard 3.0. Serial dilutions were then made of the two Citrox ® formulations using either BHI or liquid Sabouraud’s medium as the diluent. A 100 μl volume of each Citrox ® dilution was added to an equal volume of the microbial suspension, giving a range of Citrox ® concentration between 0.007% and 8% (v/v). Controls included bacterial suspensions containing no Citrox ® and un- inoculated broth. A 200 μl volume of the mixed preparations was incubated in 96-well microtitre plates for 24 hours at 37°C, under the appropriate atmospheric conditions. After incubation, the relative amount of each microbial species was estimated by measuring the turbidity of the well by spectrophotometric absorbance at 544 nm. Absorbance readings were standardised using ‘microbial-free’ Citrox ® dilutions. As recommended by Espinel-Ingroff and Cantón, 26 the minimal inhibitory concentration (MIC) value was recorded as the lowest concentration of Citrox ® that showed ≥ 80% reduction in absorbance compared to the control. Biofilm assay The concentrations of both Citrox ® formulations required to inhibit the growth of microbial biofilms were determined. Suspensions of each organism (MacFarland standard 3.0) were incubated for 24 hours at 37°C in the appropriate broth and atmospheric conditions, without agitation so as to allow the formation of biofilms. The medium was then removed by gentle aspiration and the biofilm washed with 200 μL of phosphate buffered saline (PBS) to remove planktonic cells. Fresh medium containing Citrox ® at concentrations ranging between 0.007–8% v/v was then added to the biofilm. Each antimicrobial concentration was prepared in triplicate and a control broth containing no Citrox ® wasalsoused.Biofilmswerethenincubated for a further 24 hours without movement under the same conditions as before. The medium was subsequently removed by gentle aspiration and the biofilm again washed with PBS. Fresh broth was added and the biofilms disrupted by pipetting and agitation. The turbidity of the resuspended biofilm was then observed by measuring the absorbance at a wavelength of 620 nm. A second absorbance reading (at 620 nm) was taken after a further incubation over 6 hours. The relative growth of the microorganisms was then determined by the change in absorbance over this 6 hours period. The mean value was calculated from triplicate results and the MIC recorded as the lowest concentration of Citrox ® that demonstrated a ≥ 80% reduction in absorbance compared to the control. Results Planktonic assay The MICs of each of the two Citrox ® formulations against the 14 microorganisms are shown in Table 2 and Figure 1. The planktonic growth of all of the microorganisms studied was inhibited byCitrox ® BC30. The growth of most of the species did not appear to be significantly inhibited by Citrox ® MDC30, even when it was present at the highest concentration 8% (v/v) used in this study. The MIC for each microorganism was lower with BC30 than MDC30, with the exception of Porphyromonas gingivalis for which the MIC of both formulations was the same at 2% (v/v). Overall, this suggested that BC30 was more effective than MDC30 at inhibiting microbial growth. Furthermore, as can be seen in Figure 1, BC30 inhibited the growth of each microorganism at concentrations of between 1‑2% (v/v). Biofilm assay The MIC values recorded in biofilm assay for both formulations are shown in Table 3 and Figure 2. In general the BC30 formulation was more effective at inhibiting the growth of microorganisms. One notable exception was that MDC30 appeared to be more effective against C. albicans and C. dubliniensis biofilms clinical Fig. 1 Relative growth in broth of planktonic forms of the 14 test strains in the presence of varying concentrations of Citrox®BC 30 and MDC 30ig. 1 Relative growth in broth of planktonic forms of the 14 test strains in the presence of varying concentrations of Citrox®BC 30 and MDC 30