Microsoft word - assaying bacterial survival in vivo (insects).docx
Assaying Bacterial Survival In vivo
Working protocol from Eleanor R. Haine, Department of Animal and Plant Sciences. University of Sheffield, Western Bank, Sheffield, S10 2TN. [email protected] Overview: This assay measures how quickly insects can clear pathogens from their haemolymph. Each insect is challenged with a dose of antibiotic resistant bacteria. At regular time-intervals, a subset of individuals is perfusion-bled and the number of CFUs still in the haemolymph is determined by plating out on LB agar containing antibiotics. The example protocol involves challenging adult Tenebrio molitor with Staphylococcus aureus, and should be adjusted to allow for insects of different sizes. Bear in mind that in my paper (Haine et al. submitted) 99% of a dose of 106 bacteria were gone within an hour after injection but a few were still hanging around 28 days later – adjust your protocol and dose depending on which time-points you are interested in. For example, if you’re only interested in what is happening during the first hour you could probably get away with a smaller dose of bacteria but if you want to detect bacterial growth at 14 days you’ll have to challenge them with a very large dose. Materials: tetracycline resistant Staphylococcus aureus LB broth LB agar w/ tetracycline and amphotericin B Special lab equipment needed: Incubator with shaker Ice Methods Bacterial challenges Pick one colony of tetracycline resistant Staphylococcus aureus and grow overnight in 10 ml of LB broth at 30C with shaking at 150 rpm (approximately 108 CFU ml-1, calculated from trial runs). Challenge all insects using this one overnight bacterial culture. Prepare all experimental animals by chilling on ice for 5 minutes, then inject 5 µl of S. aureus cells (straight from broth or suspended in PBS) directly into each individual between its 3rd and 4th ventral abdominal sternites. Make sure all the inoculum enters the beetle’s haemocoel. Any beetles from which haemolymph leaks out should be discarded from the experiment. After injection return beetles to standard conditions, except ca. 10 individuals. Bleed these individuals immediately after injection (i.e. 1 minute after injection) and determine CFU to calculate the average inoculums received per beetle. Measuring S. aureus survival Assay the speed of bacterial clearance by bleeding the beetles at a number of time intervals after injection. Chill each beetle on ice, and do a perfusion bleed (with 1ml PBS), collecting the perfused haemolymph into an eppendorf tube on ice. CFU per beetle is determined by plating haemolymph on LB agar containing tetracycline and amphotericin B to select for those bacteria that were injected and limit contamination from beetle cuticle or gut contents. Spread 50µl of perfusion bleed onto three plates for each beetle using glass beads and incubate for 48 hours at 30C. Count the number of colonies for each plate and take a mean from the three plates to calculate the number of
CFUs per beetle. You probably need to dilute haemolymph collected during the first hour by 10-2 – 10-4 before plating out to achieve a countable number. Calculate the number of bacteria in the total perfusion bleed and express this as a proportion of the number of bacteria that were originally injected into each individual. References Haine, E.R., Moret, Y., Siva-Jothy, M. & Rolff, J. Preventing the evolution of resistance: insect ‘antibiotic therapy’. In preparation.
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