# Antibacterial Activity of Crude Extract and Purified Acetogenins from _Amona muricata_ Seeds
## 2 Materials and Methods
### Antibacterial Assays of Crude Extract and Purified Acetogenins
Three aliquots (1 mL) of each strain were prepared and distributed as follows: untreated cells stained with 10 \(\mu\)L 3,3'-diethyloxacarbocyanine iodine (DiOC\({}_{2}\)(3)), cells treated with CE (400 \(\mu\)g/mL) and stained with 10 \(\mu\)L DiOC\({}_{2}\)(3), and cells treated with P-ACGs (400 \(\mu\)g/mL) and stained with 10 \(\mu\)L DiOC\({}_{2}\)(3). All cells were incubated at room temperature for 15 min. The membrane potential of the cell suspensions was evaluated utilizing a flow cytometer (AttuneTM, Applied Biosystems, Waltham, MA, USA) and BacLightTM Bacterial Membrane Potential Kit (B34950; Molecular Probes, Eugene, OR, USA), according to the supplier. Each staining was prepared in duplicate. The negative control consisted of an inactivated cell suspension depolarized with ten \(\mu\)L of 3,3'-carbonyl cyanide-m-chlorophenylhydrazone (CCCP). An unstained cell suspension represents a positive control. Each sample was placed in the cytometer, starting with the positive control, to adjust the detection threshold, followed by the depolarized solution and the samples. The samples were excited with a 488 nm laser, and the fluorescence emitted in the red and green channels was recorded. Forward and side scatter and fluorescence with logarithmic signal amplification were recorded. The membrane potential of the cells treated with CE and P-ACGs was assessed and compared with cells that were not treated (positive control) and depolarized cells. The magnitude of membrane potential was measured through the red: green fluorescence ratio using density plots obtained by flow cytometry. Lethality and Sublethal Injury on Enterococcus faecalis, Listeria monocytogenes, and Escherichia coli Caused by the Crude Extract and Purified Acetogenins  
The pour-plate method assessed bacterial lethality and sub-lethal injury by serial dilution [27]. Nutrient broth (200 mL) with 10 mL of cell suspension (1 \(\times\) 10\({}^{6}\) CFU/mL) adjusted to the McFarland scale (0.5 OD) was added with 100 \(\mu\)g/mL of CE or P-ACGs. The positive control consisted of cell suspension without extract. All samples were incubatedat 37 \({}^{\circ}\)C for 15 min. Subsequently, 1 mL of each treatment was mixed with 9 mL of sterile saline solution (0.85% \(w/v\)) and homogenized. Serial dilutions (up to 10\({}^{-7}\)) were prepared, and 1 mL was taken and plated into nutrient agar using a plate pouring. Each bacterial strain was subjected to this process with each treatment, and the results were expressed as log CFU/mL. Lethality was measured by the difference between log CFU/mL colony counts in the treated samples (N), and the colony counts in the control positive without the extracts (No) (Equation (5)). The difference between the CFU/mL of the treated samples and the control positive (No) samples was used to calculate the sub-lethal injury percentage (Equation (6)). \[\text{Lethality }(\text{Log CFU}/\text{mL})=\text{ Log }(\text{No}-\text{N}) \tag{5}\]  
\[\text{Sub}-\text{lethal }(\%)=\frac{\text{No}-\text{N}}{\text{No}}\times 100 \tag{6}\]