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
, 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.

tetracycline resistant Staphylococcus aureus
LB broth
LB agar w/ tetracycline and amphotericin B
Special lab equipment needed:
Incubator with shaker
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.

Haine, E.R., Moret, Y., Siva-Jothy, M. & Rolff, J. Preventing the evolution of resistance:
insect ‘antibiotic therapy’. In preparation.


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