Mini beadbeater 16 apparatus

Wild-type (CM026), cmp1Δ, and CMP1C. neoformans strains were grown in YPD broth at 30°C in a shaking incubator (220 rpm) for 24 h, centrifuged, and washed twice in phosphate-buffered saline (PBS). The cells were resuspended in PBS and quantified using a T4 cell counter (Nexcelom). Equal numbers of female and male CD-1 mice (Charles River Laboratories) were infected with approximately 5 × 10⁴ CFU per mouse via intranasal aspiration while under isoflurane anesthesia. Mice were monitored daily and observed for acute and chronic adverse symptoms. Mice were sacrificed on day 14. The brain and left lung were homogenized in 1 ml PBS for 25 s using two steel beads and a Mini-Beadbeater 16 apparatus (Biospec Products). The homogenized tissues were serially diluted, and 100 μl from each dilution was plated onto YPD containing 100 μg/ml chloramphenicol. The plates were incubated for 3 days at 30°C. Colonies were counted, and the tissue burden (CFU per gram of tissue) was determined. Fungal burden data were log₁₀ transformed and evaluated using t tests for unpaired means (Prism software, v9.1.0; GraphPad Software). A P value of ≤0.05 was considered statistically significant.

C. neoformans strain H99 was grown in yeast extract-peptone-dextrose broth at 30°C on a shaker (220 rpm) for 24 h, centrifuged (1,980 relative centrifugal force), and washed twice in phosphate-buffered saline (PBS), resuspended in PBS, and quantified by hemacytometric count. Male CD-1 mice were infected with ∼5 × 10⁴ CFU per mouse via lateral tail vein injection of 100 μl. Mice were weighed, and treatment was begun within 1 h after infection. Treatments were administered daily for 7 days. Mice were weighed daily and observed for acute and chronic adverse symptoms. Mice were sacrificed on day 8, and the brain and left lung were homogenized and cultured for quantitative determination of the tissue burden (number of CFU per gram of tissue). Tissues were homogenized for 25 s in 1 ml phosphate-buffered saline using two 6.5-mm steel beads and a Mini-Beadbeater 16 apparatus (Biospec Products, Inc., Bartlesville, OK) and serially diluted in 10-fold steps. Aliquots (100 μl) of homogenate were plated and incubated for 3 to 7 days at 37°C. Fungal burden data were log₁₀ transformed and evaluated using Kruskal-Wallis tests with Dunn's multiple-comparison test for post hoc analysis (Prism software, v5; GraphPad Software, Inc., San Diego, CA). A P value of ≤0.05 was considered statistically significant.