The fly colonies of Lucilia cuprina, Sarcophaga peregrina and Musca domestica were maintained in the insectarium of Medical Entomology Unit, Institute for Medical Research (IMR), Kuala Lumpur under 12:12 h of light dark cycle at 22 ± 2 °C and 77.0 ± 2.53% humidity with continuous supply of water and granular sugar. Female flies were provided with raw cow liver (L. cuprina), raw cow lung (S. peregrina) or moistened mouse pellet (M. domestica) for oviposition. The resultant eggs were transferred onto fresh pieces of raw cow liver and mouse pellet (L. cuprina), raw cow lung (S. peregrina) or moistened mouse pellet (M. domestica) in clean containers and the hatched larvae were constantly supplied with fresh raw cow liver (L. cuprina) or raw cow lung (S. peregrina) and water for development into late second-instar larvae.
Staphylococcus aureus ATCC 25923, Methicillin-resistant Staphylococcus aureus (MRSA S914, a clinical isolate), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were kind gifts from the Bacteriology Unit, IMR, Kuala Lumpur. These bacterial cultures were maintained on blood agar (BA). All work pertaining to the handling of bacterial cultures were performed in a EuroClone® BioAir® Microbiological Safety Cabinet Class II type A2.
All chemicals were purchased from Bio-Basic, Canada and Oxoid Ltd (BioFocus Saintifik Sdn.Bhd).
Production of larval extract
The production of larval extract was performed according to the published protocols by Teh et al.  with slight modifications. Approximately 200 unsterile, 2 to 3 days-old fly larvae were collected from cow livers, cow lungs or moistened mouse pellet and transferred into a clean, disinfected 50 ml washing tube. The unsterile larvae were washed with 40 ml of 70% ethyl alcohol and rinsed three times with sterile distilled water. Washed larvae were blot-dried with sterile paper towels and transferred into another clean, disinfected 50 ml washing tube.
Larvae were homogenised with absolute methanol (200 larvae/ 100 ml methanol). The homogenate was then transferred into clean, disinfected 50 ml centrifuge tubes and centrifuged at 4000 x g for 30 min (Eppendorf® Centrifuge 5810R). The resultant yellowish supernatant was collected and transferred into clean, disinfected glass vials. Lastly, the supernatant was concentrated using a centrifugal vacuum concentrator (Genevac miVac Quattro Concentrator) to remove methanol. The vacuum-concentrated product, i.e., the larval extract was weighed before being kept at -70 °C. Prior to antibacterial assay, 200 mg larval extract was re-suspended in 1 ml sterile distilled water and filter-sterilised with Minisart® cellulose acetate membrane syringe filter with a pore size of 0.2 μm.
Preparation of bacterial suspension
Bacteria stock cultures (S. aureus, MRSA, P. aeruginosa and E. coli) were sub-cultured onto BA plates and incubated overnight at 37 °C. The next day, three to four discrete bacterial colonies with similar morphology were inoculated into 10 ml sterile Mueller Hinton broth (MHB) and incubated overnight at 37 °C. The overnight bacterial suspensions were adjusted to 0.5 McFarland Standard with sterile MHB broth. To aid comparison, the adjustment of bacterial suspensions to the density of the 0.5 McFarland Standard was done against a white background with contrasting black lines.
Preparation of resazurin solution
Resazurin solution was prepared by dissolving 337.5 mg of resazurin powder in 50 ml sterile distilled water in a disinfected beaker. A sterile vortex mixer was used to mix the solution for 1 h to ensure homogeneity. The preparation procedures were performed in dark and the resazurin solution was then kept in a brown bottle to prevent exposure to light since it is sensitive to light.
Resazurin-based turbidometric assay and Minimum Inhibitory Concentration (MIC) determination
The resazurin-based turbidometric (TB) assay was adopted to demonstrate the inhibition effects of larval extract of S. peregrina and M. domestica against S. aureus, MRSA, P. aeruginosa and E. coli. The larval extract of L. cuprina was included into the study as another positive control in addition to standard antibiotics since its inhibitory effects had been demonstrated previously by Teh et al.  and therefore can validate and corroborate the feasibility of this assay. Broth microdilutions were performed precisely according to the Clinical and Laboratory Standards Institute (CLSI) protocol.
In a 96-well round-bottom microtiter plate, for each bacteria culture, the assay composed of one vertical row of broth sterility control, 3 vertical rows of larval extract sterility control (1 row each for L. cuprina, S. peregrina and M. domestica), 1 vertical row of growth control, 1 vertical row of antibiotic control and lastly, 3 vertical rows of larval extract test sample (1 row each for L. cuprina, S. peregrina and M. domestica). All eight wells in a vertical row were filled with 100 ul MHB. The first well of each vertical row contained 100 ul of sterile distilled water, larval extract of 200 mg ml-1, sterile distilled water, chloramphenicol (for S. aureus and MRSA) or gentamicin (for P. aeruginosa and E.coli) of 100 mg ml-1 and larval extract of 200 mg ml-1 for broth sterility control, larval extract sterility controls, growth control, antibiotic control and larval extract test samples, respectively. Subsequently, the mixture in the first well of each vertical row was mixed thoroughly. Then, a separate and sterile pipette was used to transfer 100 μl of mixture in the first well into the second well (2-2), and mixed thoroughly. Again, 100 μl of the mixture was transferred from the second well into the third well (2-3) and mixed thoroughly. This serial dilution was continued to the eighth well (2-8). Lastly, 100 μl was removed from the eighth well and discarded. The final concentration of antibiotics and larval extract was now one-half of the original concentration in each well.
Then, 5 μl of diluted bacterial suspension (1.5 x 106 cell/ml) was added into all wells (except the broth sterility and larval extract sterility control column) and mixed thoroughly. Microdilution was performed in triplicates for each bacterial species. After an overnight incubation at 37 °C, 5 ul resazurin (6.75 mg ml-1) was added to all wells and incubated at 37 °C for another 4 h. Changes of color was observed and recorded. The lowest concentration prior to colour change was considered as the Minimum Inhibitory Concentration (MIC).
Determination of antibacterial properties of larval extract
In order to elucidate the antibacterial properties (bactericidal or bacteriostatic) of larval extracts, a loopful of aliquots from the MIC wells was transferred onto brain heart infusion agar (BHIA) and incubated overnight. If bacteria failed to resume growth on BHIA after an overnight incubation, the larval extract was considered to be bactericidal, otherwise, it was bacteriostatic.
Gas Chromatography-Mass Spectrometry (GC-MS)
To determine the chemical profile of larval extracts, 1.0 μl of methanol extract of L. cuprina, S. peregrina and M. domestica larvae (1 mg/ml) was injected into a gas chromatography system (Agilent 7890A) coupled with an inert mass spectrometer (Agilent 5975C) with triple-axis detector (quadrupole). The separation of larval extract was achieved using a DB5-MS UI capillary column (30 m x 0.25 mm x 0.25 μm; 5% polydimethylsiloxane) via an autosampler (CTC Analytics) in splitless mode. Helium was used as the carrier gas with a linear velocity of 1 ml/min. The injector temperature was set at 230 °C and oven temperature was kept at 70 °C for 2 min and then increased to 270 °C at 20 °C/min.