How can Cyphox be cultured for ticks? - briefly
Cyphox can be propagated in Ixodes‑derived cell cultures using L‑15 medium supplemented with 10 % fetal bovine serum, antibiotics, and incubated at 28 °C with 5 % CO₂. Maintain subcultures every 7–10 days to preserve parasite viability for tick infection studies.
How can Cyphox be cultured for ticks? - in detail
Culturing Cyphox for use in tick studies requires a sterile environment, defined growth medium, and precise temperature control. Begin by preparing a nutrient broth composed of 0.5 % yeast extract, 0.2 % casein hydrolysate, 0.1 % glucose, and 0.05 % sodium chloride, adjusted to pH 7.2. Autoclave the medium at 121 °C for 15 minutes, then cool to room temperature under a laminar flow hood.
Inoculate the cooled broth with a single colony of Cyphox from a fresh agar plate. Use a calibrated inoculating loop to transfer the colony, ensuring the loop is flame‑sterilized before and after each transfer. Incubate the culture at 28 °C with continuous shaking at 150 rpm. Monitor optical density at 600 nm; growth reaches the exponential phase between OD 0.3 and 0.6, typically after 12–18 hours.
For large‑scale production, scale up using a 2‑liter fermenter with the same medium composition. Maintain dissolved oxygen above 30 % saturation and keep the pH within 7.0–7.4 using automatic titration with 0.5 M NaOH or HCl. Harvest cells by centrifugation at 4,000 g for 10 minutes at 4 °C. Resuspend the pellet in sterile phosphate‑buffered saline (PBS) to a concentration of 10⁸ cells ml⁻¹, suitable for tick inoculation.
Quality control steps include:
- Gram stain and microscopy to confirm morphology.
- PCR amplification of species‑specific markers to verify identity.
- Viability assay by plating serial dilutions on agar and counting colony‑forming units after 48 hours.
- Absence of contaminants confirmed by growth on non‑selective media.
Store aliquots of the prepared suspension at –80 °C in cryoprotectant solution (15 % glycerol) for long‑term use. Thaw rapidly in a 37 °C water bath before each experiment, then keep on ice until applied to ticks.
When introducing the culture to ticks, surface‑sterilize the specimens with 70 % ethanol, rinse with sterile PBS, and inject 0.5 µl of the bacterial suspension into the haemocoel using a micro‑syringe. Maintain the ticks at 25 °C and 85 % relative humidity, monitoring for infection signs over 48 hours.
Adhering to these protocols ensures reproducible growth of Cyphox and reliable delivery to tick hosts for experimental purposes.