Bacterial load in infected males
To examine how fast D. melanogaster males clear the P.
entomophila infection, we infected 1-2 day old virgin males in groups
of 20 individuals as described above. We then measured bacterial load of
individual flies at 4, 8 and 24 h from the onset of the infection
treatment, randomly choosing 2 infection vials to sample at each
timepoint. We carefully removed survivors by light anaesthesia and
randomly selected 5 individuals.
Each individual fly was then placed in an Eppendorf tube containing
small glass beads and 100μL of 70% ethanol to surface sterilize the fly
cuticle. The tube was inverted a few times to ensure proper mixing after
which the 70% ethanol was removed and replaced by 100μL of Luria broth
(LB). We then placed the Eppendorf tubes on a Precellys bead ruptor for
30 seconds at 6000rpm in order to homogenize the flies. The homogenate
was then serially diluted to obtain concentrations of 1:10, 1:100,
1:1000, 1:10,000 and 1:100,000. We plated 3 μL of each of these
dilutions in 5 replicates on a single LB plate containing 1% milk. The
plates were left for 50 hours at room temperature and colonies from each
dilution and replicate were counted. For each dilution and time point
combination, we calculated an average count of the number of colonies
for the 5 technical replicates (from each sample) followed by
calculating the total colony forming units using the formula below:
\begin{equation}
Total\ Colony\ Forming\ Units=\ Number\ of\ colonies\ for\ a\ given\ dilution\times Dilution\ factor\nonumber \\
\end{equation}