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Materials and methods

 

Three reference bacterial isolates and 18 clinical isolates of S. aureus were studied in this project. The clinical isolates were obtained from Auckland Hospital, grown overnight, mixed with glycerol and kept in -80 °C.

For each bacterial isolate, a set of 12 -15 caterpillars were infected with the bacterial solution and evaluated twice a day until they died or the assay was terminated. This was repeated on three to four separate occasions per bacterial isolate, using caterpillars from different batches. On every occasion of infection one set of caterpillars were injected with PBS and at least two sets of caterpillars were injected with reference isolates – one showing high virulence and one showing low virulence in the G. mellonella model. PBS was used as a double control, both for the physical trauma of the injection and as a control of the dilutant of the inoculums. After injection, caterpillars were placed in wells of a transparent 24-well plate with the lid taped shut and placed in a sealed plastic container to prevent escape. The plates were incubated at 37°C in the dark for approximately 100 hours.

The caterpillars were assessed using the caterpillar health index (CHI) scoring system two times per day and time and date were recorded. Dead caterpillars were immediately taken out of the assay. Four to six caterpillars per set of inoculated caterpillars and assay were further analysed for number of viable bacterial cells in a colony forming unit (CFU) assay. 

In the CFU assay, the number of colony forming units in dead caterpillars were calculated. This data reveals the concentration of viable bacterial cells in each caterpillar after death and can be used to analyse if the strains/isolates differ in the concentration of bacteria required to kill the caterpillar.

All assays were single blinded by a laboratory worker not involved in the research. After the infection, the 24-well plates were marked with a letter in exchange for the strain name. The blind key was revealed when all caterpillars were dead.


Responsible for this page: Agneta Johansson
Last updated: 05/06/13