Micro fine insulin syringe

Conidia suspensions of strain CBS144.89 (Dal), its bioluminescent KU⁸⁰ deletion strain (ΔakuB N5) (Resendiz-Sharpe et al., 2022 (link)), as well as strain NIH4215 and its bioluminescent and nmt1-complemented ΔakuB strains 2/9 and 4/11 were freshly prepared from glucose/glutamine minimal medium slopes supplemented with or without thiamine. Conidia were harvested in PBS (Gibco) containing 0.01% Tween 20, washed twice and resuspended in PBS. Larvae of the greater wax moth Galleria mellonella (Waxworms Ltd - Sheffield, UK) were kept at 15°C for up to one week before starting the infection studies. To compare the virulence of wild-type strains and bioluminescent ΔakuB transformants, groups of 10 larvae weighing 200-300 mg were inoculated each with 50 µl PBS without (mock control) or with 1 × 10⁵, 2.5 × 10⁵, 5 × 10⁵ or 1 × 10⁶ conidia. Injections into the haemolymph were performed through the second pro-leg of G. mellonella larvae, using 0.5 mL BD Micro-Fine™ insulin syringes. In addition, a group of non-injected larvae served as treatment control. After infection the larvae were kept at 37°C in the dark and their survival was monitored daily for up to 6 days post-infection. The data was plotted in Kaplan-Meier survival curves and statistical analysis was performed by log-rank test using GraphPad Prism version 8.0.0 for Windows.

Galleria mellonella infections were performed as described previously (78 (link)). The wax moth Galleria mellonella was obtained from UK Waxworms, Ltd. (Sheffield, UK). C. albicans strains were grown in GYNB with or without lactate for 5 h at 37°C, and the cells were washed and resuspended in PBS. Upon arrival, the larvae were kept in wood shavings at 12°C for up to 2 weeks before the experiments. Initially, to compare killing rates for wild-type and deletion strains, groups of 15 larvae weighing 200 to 300 mg were inoculated with 1 × 10⁸, 5 × 10⁷, 5 × 10⁶, and 4 × 10⁵ cells/mL of the wild-type strain. Based on these comparisons, a dose of 2.5 × 10⁵ cells was used in subsequent experiments. Each larva was injected with 50 μL of fungal cell suspension through the second proleg, using a 0.5-mL BD Micro-Fine insulin syringe, and noninjected and PBS controls were included. The larvae were then incubated at 37°C in the dark, and survival was quantified for 10 days postinfection.