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}