**2.8 Microbiological analysis**

After treatment procedures each implant was placed in a tube containing 500 μl of phosphate buffered saline (PBS). The tubes were vortexed for 60 seconds (Vortex, Genius 3, IKA, Germany). This was done to remove the remaining bacterial cells from the implant surfaces.

From each tube, 100 μl were taken and using a 96-well microtiter plates a ten-fold dilution was performed and 30 μl of suspension from each well was then inoculated to Brucella agar plates.

The plates were placed in anaerobic conditions and after 72 hours and the colony forming units per milliliter (CFU/ml) were counted (**Figure 4**). MALDI Biotyper (Bruker Daltonics, Germany) was used to macroscopically differentiate distinctive colonies.

### **2.9 Scanning electron microscopy analysis**

Scanning electron microscopy (SEM) was performed on one randomly selected implant from each of the treatment groups and one sterile non-treated implant. The implants for SEM analysis were stored for 2 hours in 2% paraformaldehyde and, later on dehydrated in increasing concentrations of ethanol (60%, 75% and 95%), for 30 minutes in each and dried overnight. The surfaces of the prepared implants

**157**

(**Table 5**).

*Evaluation of the Antimicrobial Efficacy of Different Types of Photodynamic Therapy…*

were analyzed by SEM (Vega TS5136MM, Tescan, Brno, Czech Republic). The SEM images were taken at 1:250 magnifications under high vacuum (HiVac) with a high voltage (HV) of 30 kV. All the images were taken between the fourth and the fifth thread of the implants. As for the zirconia implants, they are non-conducting material and in order to make the samples conductive and avoid charging of the sample surface, the implants were coated with gold and palladium sputter (SC7620 Mini

The differences between the groups for each bacterial species separately and for the total count of bacteria, were compared by analysis of variance test (ANOVA) and Tukey test, as a post hoc. The level of significance was set at 5%. The statistical package SAS system for Windows (Release 8.02, SAS Institute Inc., Cary, NC, USA)

To determine the difference among the groups and between the two types of

The comparison between the two types of implants: titanium and zirconia, regardless of the study groups, showed that there was a significantly lower number of bacteria on zirconia implants for all three types of bacteria separately, as well as

For the comparison among the study groups regardless of the type of implant, Tukey test was applied. Regarding the total number of bacteria, the least bacteria were found in PDT1 and PDT2. These two groups were followed by PDT3 and PC without significant difference among them. The negative control group (NC) as expected, had the largest number of bacteria compared to the other groups. The

The total number of bacteria for every group and both implant types are shown

There were statistically significant differences among the groups for each of the bacteria separately and also for the total number of bacteria (p = 0.0022). These results are presented in **Table 5** in logarithmic form. Regarding the total number of bacteria, the largest reduction was observed in the PDT1 (98.3%) and PDT2 (97.8%) groups. These two groups had statistically significant difference when compared to NC (p < 0.05). In the PDT3 group there was a 68.7% bacterial reduction, without statistically significant difference when compared to NC

When each bacteria was compared separately, the PDT1 and PDT2 groups also showed the largest bacterial reduction. PDT1 group, was significantly more effective in the eliminating *A. actinomycetemcomitans* and *P. gingivalis* (p < 0.05).

same results were obtained for the number of each bacteria separately.

for each of the bacteria separately are shown in **Figures 6**–**8**.

in **Figure 5** in schematic form. The difference between zirconia implants and titanium implants was not the same for all groups. The smallest difference between both types of implants in the number of bacteria was for the control group. The impact was almost the same for PDT1, PDT2, PC and TB, while the largest difference between titanium and zirconia implants were in the PDT3 group. The results

implants, multivariate analysis of variance test was applied.

for the total number of bacteria (**Table 4**).

*DOI: http://dx.doi.org/10.5772/intechopen.94268*

Sputter Coater, Quorum Technologies Ltd., UK).

**2.10 Statistical analysis**

**3.1 Titanium implants**

was used.

**3. Results**

**Figure 4.** *Visible colonies of bacteria on Brucella agar plate.*

*Evaluation of the Antimicrobial Efficacy of Different Types of Photodynamic Therapy… DOI: http://dx.doi.org/10.5772/intechopen.94268*

were analyzed by SEM (Vega TS5136MM, Tescan, Brno, Czech Republic). The SEM images were taken at 1:250 magnifications under high vacuum (HiVac) with a high voltage (HV) of 30 kV. All the images were taken between the fourth and the fifth thread of the implants. As for the zirconia implants, they are non-conducting material and in order to make the samples conductive and avoid charging of the sample surface, the implants were coated with gold and palladium sputter (SC7620 Mini Sputter Coater, Quorum Technologies Ltd., UK).

#### **2.10 Statistical analysis**

The differences between the groups for each bacterial species separately and for the total count of bacteria, were compared by analysis of variance test (ANOVA) and Tukey test, as a post hoc. The level of significance was set at 5%. The statistical package SAS system for Windows (Release 8.02, SAS Institute Inc., Cary, NC, USA) was used.
