Fly survival was monitored and recorded from 12 to 72 hours post inoculation.
Survival curves were generated by the Kaplan-Meier method, and statistical significance was calculated by log-rank test using Prism 5 (GraphPad Software, Inc.). Bacterial in vitro growth curve Overnight bacterial cultures were diluted (1:1000) in fresh BHI broth or M9 minimal salt medium (BD Biosciences), with 200μl loaded onto a 96-well plate. Each well was covered with 50 μl of mineral oil to prevent evaporation. The growth curves of bacterial cultures at 25°C, which mimics the temperature Vactosertib datasheet inside fly body, were monitored photometrically by reading the optical density at 600nm using an automatic optical density measuring
machine (1420 Multilabel Counter VICTOR, Perkin Elmer). Bacterial in vivo growth inside flies Bacterial replication was monitored throughout the fly pricking experiments, and only the live flies Smoothened Agonist purchase were assessed. In order to enumerate viable bacteria in the whole fly at 1, 6, 18, and 24 hours post infection, 8 infected flies were harvested, and the whole flies were homogenized using pestles (DiaMed), and the bacterial number per fly was enumerated. In order to enumerate the bacteria present in specific body parts (i.e. crop, head, leg, and wing), 8–10 infected flies were harvested and dissected at 18 hours post infection, with the specific body parts collected RAD001 into 100μl phosphate buffered saline (PBS) followed by homogenization. The quantitative bacterial counts in the different body parts of each fly were enumerated. For both the whole fly and body part harvesting,
the homogenate was re-suspended in 1 ml of PBS, and 100μl of 10-fold serial Histidine ammonia-lyase dilutions were plated onto tryptic soy agar (TSA) with ampicillin (50μg/ml). Colonies were counted following overnight incubation at 37°C. The Mann–Whitney test was performed to determine significant differences between the different strains. For microscopic examination of the whole fly, the infected flies at 18 hours post infection were fixed in 10% neutral-buffered formalin and sent to the Histopathology Laboratory at the Faculty of Veterinary Medicine, University of Calgary, for processing, sectioning, and Gram staining. RNA isolation and reverse transcription For bacterial virulence gene expression in vitro, 0.5-ml of bacterial culture at the mid-log phase (OD600 ~0.6) and the stationary phase (OD600 ~ 4.5 for CMRSA2 and CMRSA6, and OD600 ~ 5.0 for USA300, USA400 and M92, based on the bacterial growth curve measurements for each strain) were aliquoted. The total RNA was extracted using TRIzol (Invitrogen). For host antimicrobial peptide (AMP) gene expression or in vivo bacterial virulence gene expression, total RNA from five flies chosen randomly at 6, 18, and 24 hours post-infection were extracted using TRIzol, as previously described .