Insulin 29g u 100 needle

In order to determine the efficacy of currently used antifungal agents, G. mellonella were infected with 10 mg/larvae of F. senegalensis as described above. On 4 h, 24 h, and 52 h after infection, 15 larvae per experiment were treated with either 1 mg/kg amphotericin B (Fungizone, Bristol Myers Squibb, Utrecht, The Netherlands), 5.7 mg/kg itraconazole (Janssen Pharmaceuticals, Beerse, Belgium), 7.14 mg/kg terbinafine (Sigma, Missouri, USA), or PBS. Antifungal agents were administered in a 20 µl volume, each time via a different proleg with an insulin 29 G U-100 needle (BD Diagnostics, Sparsk, USA). Larval survival was monitored for 10 days. Every treatment was replicated 3 times.

To generate hyphal fragments that could be injected into G. mellonella larvae, mycelia from F. senegalensis were obtained from 2-week old cultures grown on Sabouraud agar plates and inoculated in RPMI 1640 culture medium supplemented with L-glutamine (0.3 g/l; Capricorn-Scientific, Germany), 20 m m mopholinepropanesulfonic acid (MOPS; Sigma, USA), and chloramphenicol (100 mg/l; Oxoid, Basingstroke, UK). After 2 weeks of incubation at 37°C, hyphae were collected by filtering them through a 0.22 µm filter (Nalgene, Abcoude, The Netherlands) and washed with phosphate-buffered saline (PBS; Gibco, USA). To obtain hyphal fragments, the washed hyphae were sonicated for 10 s at 20 µm (Soniprep, Beun de Ronde, The Netherlands), as previously done for M. mycetomatis.^{8 (link)} To establish the lethal dose, different inocula were prepared in PBS. These were: 10 mg/larvae, 4 mg/larvae, 0.4 mg/larvae, and 0.04 mg/larvae. A total of 40 µl was injected into the last left pro-leg of the larvae with an insulin 29 G U-100 needle (BD Diagnostics, Sparsk, USA). Uninfected controls were injected with 40 µl PBS. A total of 15 larvae per group were used, and every test was replicated three times. Larvae were placed in a petridish containing Whattman paper and incubated at 37°C in a normal incubator. Survival was monitored for 10 days.

Wax moth larvae killing assays were carried out as described previously.29 (link) A total of 20 larvae were used for each testing group. Larvae were first infected each strain with different concentrations of conidia (5×10⁶, 2.5×10⁶, 1.25×10⁶, 5×10⁵ and 1×10⁵ conidia per larva). Inoculation was performed by injecting 50 μL of the fungal suspension at the last left pro-leg with an insulin 29G U-100 needle (BD diagnostics, Sparks, USA). For control larvae, larvae were also pricked with the needle and injected with PBS. Larvae were checked daily for survival for 7 days at 37 °C.