# Antibacterialativity of _Lysimachia nummularia_ L. in Oro-Dental Diseases
## 2 Materials and Methods
### Testing the Bacterial Sensitivity to the Extracts Taken in the Study Compared to the Antibiotics  
An antibiogram is a laboratory qualitative test that determines the sensitivity of a bacterial isolate to different antibiotics and chemotherapeutics. It involves growing a sample of the bacteria on a culture plate and then exposing the bacteria to various antibiotics and chemotherapeutics. The growth of the bacteria is then observed to determine which antibiotics are effective in inhibiting or killing the bacteria [20,21].  
To test the sensitivity of bacteria to antibiotics, the effusimetric method is used, which is a qualitative method.  
The principle of the method aims at seeding a bacterial suspension to be tested on the surface of a gelatinous culture medium, followed by the subsequent deposition of microcompresses with different antibiotics or chemotherapeutics. Antibacterial substances diffuse into the culture medium, showing a constant decrease in the concentration gradient from the edge of the antibiotic disk to the periphery. After a 24 h incubation at 37 \({}^{\circ}\)C, the interpretation is made, which consists of measuring the zone of inhibition, in which the bacteria did not develop, the dimensions of this zone being directly proportional to the sensitivity of the bacteria to the respective antibiotic.  
Given the fact that in the present study we wanted to assess the level of sensitivity of some bacterial strains to the obtained extracts, we adapted the effusimetric method, using the same principle but replacing the antibiotic tablets with sterile filter paper rounds, with the same diameter as of the antibiotic tablets, impregnated in the test solution; we quantified the volume of saturating solution and found that the filter paper washer is saturated with 10 uL.  
The _interpretation of results_ is performed by measuring the diameter of the zone of inhibition of the development of bacterial colonies around the filter paper washers, expressed in mm.  
If the medium is transparent, the reading is performed directly on the bottom of the plate, in reflected light, and if the media of the plates is agar-blood, the diameter measurement is made on the surface of the medium.  
The expression of the results is conducted by relating the size of the zone of inhibition to the standards of critical diameters for different antibiotics, which allows the definition of strains as sensitive, intermediate, and resistant [22, 23, 24]. The bacteria that we had the right to test were grown on various culture media; as a positive control, antibiotics and antifungals were used, and as a negative control, distilled water was used (Table 1).  
\begin{table}
\begin{tabular}{c c c c} \hline
**Wild Bacterial Species** & **Test Environment** & **Positive Control—Antibiotics/Antimycotics** & **Negative Witness** \\ \hline _Streptococcus mitis_ & Blood agar & Penicillin G (P) & Distilled water \\ \hline _Staphylococcus aureus_ & Blood agar & Penicillin G (P) & Distilled water \\ \hline _Enterococcus_ & Mueller–Hinton & Ampicillin (AMP) & Distilled water \\ \hline _Escherichia coli_ & Mueller–Hinton & Gentamicin (CN) & Distilled water \\ \hline _Pseudomonas aeruginosa_ & Mueller–Hinton & Gentamicin (CN) & Distilled water \\ \hline _Candida albicans_ & Sabouraud & Fluconazole (FCA), & Distilled water \\  & & Voriconazole (VOR) & Distilled water \\ \hline \end{tabular}
\end{table}
Table 1: Bacterial species and antibiotics/chemotherapy used.  
The plates were incubated for 24 h at 37 \({}^{\circ}\)C, after which the results were read by measuring the zones of inhibition in millimeters for each reference bacterial species; three readings were taken, and the mean and standard deviation (SD) were obtained.