## 3 Results and Discussion
The results show that there is a significant difference among the final RSE-formazan content of these three methods (\(p<0.0001\)). The isopropanol-dissolving RSE-formazan showed the highest OD\({}_{570}\) values over acidic isopropanol dissolving and after column chromatography-filtered RSE-formazan solutions. The final RSE-formazan content from the column chromatography-filtered method is significantly lower than the final RSE-formazan content from the common method. Then, we used TLC plates to verify the components of the final RSE-formazan solutions from the common method and the column chromatography method. In Figure 5, the TLC plate shows that there is a yellow dot at the bottom of A (the acidic isopropanol RSE-formazan) (R\({}_{f}=0.00\)). However, in the column chromatography-purified RSE-formazan (B), there are no visible component residues in the bottom. Therefore, the TLC plate results support the hypothesis that coloured components of BHI broth, RSE, and bacterial cells were adsorbed by the silica gel during the eluent procedure (Figure 5). Figure 3: The optical density results of the BHI broth before (isopropanol dissolving) and after (column chromatography) filtering, and _S. aureus_ suspension. Bacterial broth before filtering: _S. aureus_ suspension dissolved in isopropanol; BHI broth before filtering: pure BHI broth dissolved in isopropanol; bacterial broth after filtering: _S. aureus_ suspension obtained after column chromatography filtration; BHI broth after filtering: pure BHI broth obtained after column chromatography filtration. Results are expressed as the mean \(\pm\) SD. Quadruple asterisks (****) indicate \(p<0.0001\). ns indicates \(p>0.05\). Figure 4: The final formazan content of NZ ‘Hass’ ripe avocado seed extract treated in the _S. aureus_ suspension (RSE-formazan) using three dissolving methods. Common method: acidic isopropanol dissolving method; before filtering: isopropanol dissolving method; after filtering: after column chromatography filtering method. Results are expressed as the mean \(\pm\) SD. Quadruple asterisks (*******) indicate \(p\) < 0.0001. ns indicates \(p\) > 0.05. Figure 5: The TLC plate of formazan that was produced from NZ ‘Hass’ ripe avocado seed extract treated in the _S. aureus_ suspension. 1 indicates common method: The formazan dissolved using the acidic isopropanol dissolving method; 2 indicates after filtering: The formazan dissolved using the column chromatography filtering method; 3 indicates standard: The formazan standard solution. NZ 'Hass' avocado ripe seed extract has been reported to contain a high phenolic content [25]. Additionally, several phytochemicalen studies showed that phenolic extracts contain antibacterial activities [27; 28; 29]. TLC is a well-established analytical technique to be used to separate and identify the components in complex plant extracts [30]. It is a valuable method to guide the column chromatography by providing mobility, purity, and polarity information on the components in plant extracts [31]. We considered the polarity character of the ripe avocado seed crude extracts, IPA, and formazan. The polarity of these three materials is: formazan < IPA < avocado ripe seed crude extracts (phenolic compounds). Current TLC results (Figure 5) show that IPA can selectively partition different compounds with different polarities, allowing them to be separated on the TLC plate. The results from the TLC plate suggest that IPA is a suitable solvent for separating the formazan and RSE (phenolic compounds) in column chromatography. The RSE (phenolic compounds) was strongly adsorbed to the stationary phase (silica gel) in the column. As the first column chromatography and MTT colorimetric combination method, it removed the background colour (broth and plant extracts), and bacterial cell interferences before formazan microplate testing. These interferences could affect the formazan measurement and lead to false-positive or false-negative results.