Crithidia mellificae strain SF - Microscopy, Culturing and DNA Purification

Honey bees were collected from a San Francisco, CA (U.S.A.) colony previously identified to be Crithidia positive by microarray and PCR testing. Honey bees were immobilized by chilling at 4°C for 20 minutes, briefly washed in 70% ethanol, and decapitated prior to dissection. The SF strain was isolated from honey bee intestines dissected in a sterile environment, minced and placed in a T25 flask and cultured in BHT medium composed of Brain Heart Infusion (BHI) 28.8 g/L (DIFCO), tryptose 4.5 g/L (DIFCO), glucose 5.0 g/L, Na₂HPO₄ 0.5 g/L, KCl 0.3 g/L, hemin 1.0 mg/L, fetal bovine serum (heat inactivated) 2% v/v, pH 6.5, and containing penicillin G sodium (10⁶ units/L) and streptomycin sulfate (292 mg/L) at 27°C [101]. Free active Crithidias were observed 24 hours post inoculation. Parasites were maintained by subculture passage every 4 days; stable liquid nitrogen stocks were archived. Light microscopy of live parasites was performed using a Leica DM6000 microscope equipped with Hamamatsu C4742-95 camera and Volocity Software (PerkinElmer). Imaging fixed parasites (4% paraformaldehyde, 20 min) facilitated visualization of DAPI (4′,6-diamidino-2-phenylindole) stained nuclear and kinetoplast DNA. Images of fixed Crithidia mellificae were obtained using both the Leica DM6000 microscope and a Zeiss LSM 510-M microscope equipped with both a 63× objective numerical aperture 1.4, and a 100× objective numerical aperture 1.4.

For DNA purification, Crithidia mellificae (∼10⁶ trypanosomes/mL culture medium) were pelleted by centrifugation (800×g for 6 min) and washed with PBS prior to DNA extraction. DNA was extracted using the DNeasy Genomic DNA Extraction Kit (Qiagen) as per the manufacturer's instructions. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).