# Lactic Acid Modified Natural Rubber-Bacterial Cellulose Composites
## Materials and Methods
### Characterization of Composite Films
As water solubilized into the film and permeated through the sample material, nitrogen gas swept and transported the transmitted water vapor molecules to a calibrated infrared sensor on the other side. Thereafter, the transmission rate was determined. Biodegradation of NRBC and ANRBC composites in soil was carried out for 6 months. Each test specimen was in the form of \(5\times 5\) cm\({}^{2}\) and buried to a 10 cm soil depth in a pot under the natural soil environment, where the soil moisture was around 11-14 (% vol) and soil temperature was around 27-33 \({}^{\circ}\)C. The samples were weighed after 1, 3, and 6 months. For calculation of biodegradation, the samples were carefully taken out, washed with distilled water, and dried at 50 \({}^{\circ}\)C for 24 h and then weighed. The biodegradation based on percent by weight of mass loss was calculated as  
\[\text{Biodegradation }(\%)=\frac{W_{1}-W_{2}}{W_{1}}\times 100 \tag{2}\]  
where \(W_{1}\) is the initial dry weight of the samples, and \(W_{2}\) is the residue dry weight of films of the samples after biodegradation in soil. Antibacterial tests of NRBC and ANRBC films against _E. coli_ and _S. aureus_ were determined by the disc diffusion method. _E. coli_ or _S. aureus_ was suspended in nutrient broth at the standardized concentration of \(\approx\)10\({}^{8}\) cells/mL prior to spreading 1 mL the prepared bacterial broth onto an agar plate. Then, the film samples were cut into 1 cm circular discs and were gently placed on the surface of an agar plate. The plates were incubated at 37 \({}^{\circ}\)C for 24 h. After that, the clear zones of growth inhibition around sample infused discs were measured. The method for cytotoxicity tests followed similar procedures to those in previous reports [13; 14]. The cytotoxicity was tested against human embryonic kidney cells (HEK 293) and human skin keratinocytes (HaCaT). The cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% l-glutamine, 100 unit/mL penicillin, 2 mg/L laclalbumin, and 100 ug/mL streptomycin in a humidified incubator with an atmosphere of 5% CO\({}_{2}\) at 37 \({}^{\circ}\)C. A film sample of 0.5 g was autoclaved at 121 \({}^{\circ}\)C for 15 min. The sterilized film was extracted by immersion in 10 mL DMEM at 37 \({}^{\circ}\)C for 24 h. Then, the extract was filtrated through a 0.45 \(\upmu\)m filter, and the concentration was adjusted to 1000 \(\upmu\)g/mL. Cells of \(1\times 10^{4}\) cells/mL were seeded into a 96-well plate of DMEM at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\). After 24 h, the DMED was replaced by 100 \(\upmu\)L/well of the extract and the cell was further incubated for another 24 h using the same conditions. A positive control was the cell cultured in DMEM. Then MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolzolium bromide) assay was carried out to determine the number of living cells. The extract was removed, and then 10 \(\upmu\)L of MTT reagent was added into each well. The incubation was performed for 4 h at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\). After that, MTT solution was removed and 150 \(\upmu\)L dimethyl sulfoxide (DMSO) was added into each well to solubilize blue formazan crystals. Colorimetric detection was done by a UV-VIS spectrophotometer (Thermo Scientific, Waltham, MA, USA) at a wavelength of 570 nm. The amount of color produced was directly proportional to the number of viable cells. The viability of cells in the extract medium was compared to that of the control medium (DMED). The percentages of cell viability was expressed as mean \(\pm\) SD (n = 3).