Ultrospec 10 spectrophotometer

Mitomycin C (Sigma, Sydney, NSW, Australia) was used to induce the putative antibacterial proteins as outlined by [46 (link)]. Single colonies of bacteria were used to inoculate 5 mL of LB (Miller, Sigma) broth, which was placed on an orbital shaker (Conco, TU 4540, Taibei, Taiwan) at 250 rpm and 30 °C overnight. Aliquots (500 µL) of the overnight culture were transferred to further inoculate 25 mL of LB broth. The culture was left to grow (~10–12 h) at 250 rpm and 30 °C until it attained turbidity. Two concentrations (1 µg/mL and 3 µg/mL) of Mitomycin C (Sigma) were independently added into the flasks, which were then positioned on an orbital platform for incubation with rotation at 40 rpm at ambient temperature (24°C). OD_600nm readings were recorded through an Ultrospec-10 spectrophotometer (Amersham Biosciences, Amersham, UK) after 2, 4, 6, and 24 h of Mitomycin C (Sigma) addition. The flasks were monitored for signs of cell lysis (clearing of the culture or accumulation of bacterial debris). The flasks without the addition of Mitomycin C served as a control. All the treatments in the experiment were technically replicated four times. OD_600nm readings at various time intervals were pooled and statistically analysed using the ANOVA (Analysis of Variance) test through the Genstat 20th edition programme.

Cultures of S. aureus (ATCC 29213) and P. aeruginosa (ATCC 27853) were prepared by inoculating Mueller-Hinton agar growth medium (Biorad) and incubated overnight at 37°C. For wound inoculation, a bacterial mixture containing both strains in equal proportions was prepared in 1X PBS (Dulbecco) by resuspending each strain at a final density of 2.5 x10⁸ CFU/ml. Bacterial concentration was determined by measuring the absorbance at 600 nm using an Ultrospec-10 spectrophotometer (Amersham Biosciences) and by colony-forming unit (CFU) counts for each strain controlled by serial 10-fold dilutions of the bacterial suspensions and plating on either MH (Mueller Hinton)/vancomycin and CNA (Columbia Naladixic Acid) selective agar plates.