3.0 Hohenstein Challenge Test (AATCC TM100)
In vitro antimicrobial activities against test microorganisms were conducted for both the negative control and nanocomposite hydrogels with phomopsidione (Rejendra et al., 2011). At the start, one square hydrogel measuring 20 mm by 20 mm was added into each flask containing nutrient broth (Merck). In the meantime, the flasks were inoculated with 100 µL of test microorganism where it was allowed to grow aerobically in the incubator shaker at 120 RPM, 37˚C for 24 hours. Then, serial dilution was done before spreading the solution on nutrient agar plates. The dilution factor of 10-2 was used for the flasks containing nanocomposite hydrogels with phomopsidione and the dilution factor of 10-7 was used for flasks containing negative control. The diluted bacterial solution was then distributed evenly on the nutrient agar (Merck) plates using an L-shaped spreader. Each test microorganism was cultured for 24 hours and counted (Liu & Kim, 2012). The Colony Forming Unit (CFU)/mL and percentage bacterial reduction for each sample was calculated using the following formulae.
\begin{equation} \%\ Bacterial\ Reduction=\frac{\text{CFU\ before\ treatment\ }\times\text{CFU\ after\ treatment\ }}{\text{CFU\ before\ treatment}}\times 100\%\nonumber \\ \end{equation}