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Assaying Bacterial Killing In vitro Working protocol from Eleanor R. Haine, Department of Animal and Plant Sciences.University of Sheffield, Western Bank, Sheffield, S10 2TN. e.haine@sheffield.ac.uk

Overview: This assay simply involves adding a known starting concentration of bacteriato haemolymph and incubating the solution over a couple of hours. After incubation the

bacteria are plated out and the number of CFUs remaining is compared to the startingconcentration. It assumes that the decrease in CFUs is proportional to the antibacterialactivity of the insects haemolymph. So far I have only tried it with S. aureus .

Materials:liquid culture of antibiotic-resistant Staphylococcus aureus

broth mediaPBSLB agar plates w/ relevant antibiotic and amphotericin B

Special lab equipment needed:Eppendorf tubesIceIncubator w/ shaker Glass beads

MethodsI developed this assay to use a very small volume of undiluted haemolymph, since thatwas all I had. You could use neat haemolymph as I did and multiply up the relative

proportions of PBS and bacteria for a larger volume. Alternatively you could use dilutehaemolymph, from a perfusion bleed for example, but you may need to play around withrelative volumes a bit to get it right. The idea is to have enough bacteria that you can seea difference, but not so many that you have to spend hours diluting the assays before

plating out.

Preparing assayCollect neat haemolymph into an eppendorf tube and freeze at 90C to rupturehaemocytes and release cell contents. Meanwhile prepare a liquid culture of antibiotic-resistant Staphylococcus aureus : pick one colony and grow overnight at 30C(approximately 108 CFU ml-1). Defrost haemolymph sample on ice. Dilute 1ulhaemolymph with 24ul sterile PBS at room temperature. Add ul of overnight culture of S.aureus , briefly vortex to mix and then incubate at 30C, with shaking at 150rpm for 2hours.

Plating out assayPrepare LB agar plates containing the relevant antibiotic and amphotericin B. Diluteassay mixture 100 times and then pipette 20ul of diluted assay solution onto an agar plateand spread evenly using glass beads. Incubate plate at 30C for 24-48 hours and thencount the number of CFUs on each plate.

Calculate killing strength

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Multiply CFU counts per plate up to total in assay, e.g. if you had 50 CFU on a platefrom plating out a volume of 20l, multiply this by 100 to account for the dilution, i.e.500 CFU. This is from 20ul from a total assay volume of 26ul, so divide 500 by 20 andmultiply it by 26: 650 CFU. Subtract this number from the starting concentration toexpress the anti- S. aureus activity of the haemolymph as the number of S. aureus CFUskilled during 2 hours of exposure to T. molitor haemolymph.

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