## 2 Materials and Methods
The method consists of placing paper disks saturated with antimicrobial agents on a lawn of bacteria, seeded on the surface of an agar medium, incubating the plate overnight, and measuring the presence or absence of a zone of inhibition around the disks [12]. In accordance with this procedure, _Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans_ were inoculated separately on Mueller-Hinton culture media [13]. The laboratory procedure was performed according to the Kirby-Bauer disk-diffusion-susceptibility-test protocol of the American Society for Microbiology. The culture media were at room temperature prior to inoculation. Sterile rayon swabs were dipped in the microbial suspension. In the case of gram-negative bacteria, the excess fluid was removed by pressing and turning the swab against the inside of the tube to avoid over-inoculation. The inoculum was evenly spread over the entire agar surface, without gaps between streaks. Three sterile paper discs containing 7 uL of each disinfectant were placed on each culture-medium surface within 15 min of inoculation. Disks were placed in close and firm contact with the agar surface, and were not moved once applied. The plates were inverted to confirm that the disks would not fall off the agar surface. The 24 h incubation at 35 \({}^{\circ}\)C had to begin within 15 min of the disk application. The results were read after 24 h. For basic research, a mix of these microorganisms was used, after detecting the susceptibility of each microorganism to the studied disinfectants. The colonies were inoculated in saline solution and standardized using the McFarland scale (0.5 McFarland = 1-2 \(\times\) 10\({}^{8}\) CFU/mL) [14]. The American Society of Microbiology protocol for obtaining the 0.5 McFarland standard consists of verifying the correct density of the turbidity standard by measuring absorbance, using a spectrophotometer with a 1 cm light path and matching cuvette. The absorbance at 625 nm must be 0.08 to 0.13 for the 0.5 McFarland standard. An equimolecular mixture was made with the three microorganisms (Figure 1) \begin{table}
\begin{tabular}{c c c c} \hline
**Microorganisms** & **Type** & **Shape** & **Frequently Found** \\ \hline _Staphylococcus aureus_ & Gram-positive bacteria & Spherical-shaped & Upper respiratory tract, skin \\ \hline _Pseudomonas aeruginosa_ & Gram-negative bacteria & Rod-shaped & Soil, water, skin \\ \hline _Candida albicans_ & Fungi & & Gastrointestinal tract, mouth \\ \hline \end{tabular}
\end{table} 
Table 3: Basic characteristics of the microorganisms. A total of 150 sterile containers of 60 mL (Nantong Bestreatm Medical Instrument Co. Ltd., Nantong, China) were prepared. Each contained 20 mL of saline solution, 20 mL of microorganism suspension, and one impression-material specimen (Figure 2). Each container was shaken well for 10 s. The impression-material specimens were removed from the microorganism suspension after 10 min. This is considered the maximum handling time in a dental office, from the beginning of the impression procedure until the final impression would be disinfected. After rinsing the specimens with water for 30 s, they were organized into three different trays. Each tray contained ten specimens of each of the five impression materials. Control samples were collected from each specimen using sterile rayon swabs, according to the recommendation of the American Society of Microbiology, pressed onto the surface, and moved in two directions (right and left) at right angles. The stick of the swab was rotated between the thumb and forefinger for 15 s. The disinfectants were applied from 15-20 cm distance until the whole surface of the specimens was equally wet, and left to act according to the manufacturer's recommendations: 5 min after spraying for Zeta 3 Soft, 3 min after spraying for Zeta 7 Spray, and 10 min of immersion for Zeta 7 Solution. After this period, the impressions were rinsed for 10 s with water.