How to cultivate entomozan from ticks? - briefly
Collect engorged ticks, surface‑sterilize them, and inoculate a nutrient agar enriched with insect hemolymph; incubate at 25 °C with high humidity for 5–7 days to induce entomozan growth. Sub‑culture the resulting colonies weekly onto fresh medium to sustain the culture.
How to cultivate entomozan from ticks? - in detail
Cultivating entomozan from tick sources requires a controlled laboratory environment, precise aseptic techniques, and compliance with biosafety regulations.
The process begins with specimen acquisition. Collect adult or nymphal ticks from a verified population, preferably using CO₂ traps or flagging methods. Place specimens in a refrigerated container (4 °C) and transport them to the laboratory within 24 hours to preserve viability.
Preparation of the inoculum involves surface sterilization and tissue extraction. Immerse each tick in 70 % ethanol for 30 seconds, followed by three washes in sterile phosphate‑buffered saline. Dissect the salivary glands or midgut, homogenize the tissue in a defined volume of sterile broth (e.g., Luria‑Bertani or a specialized insect cell medium), and filter the suspension through a 0.45 µm membrane to remove debris.
Incubation proceeds under the following conditions:
- Temperature: 27 °C ± 1 °C
- Shaking speed: 120 rpm (if using liquid culture)
- Aeration: continuous airflow or intermittent venting
- Duration: 48–72 hours, monitoring turbidity and optical density at 600 nm
During incubation, supplement the medium with trace elements (iron, magnesium) and a carbon source (glucose or sucrose) to support metabolic activity. Periodically sample the culture for microscopic examination and quantitative PCR to confirm entomozan expression.
Harvest the product by centrifugation at 5,000 × g for 10 minutes. Resuspend the pellet in a chilled buffer (50 mM Tris‑HCl, pH 7.5) and perform a series of purification steps: ion‑exchange chromatography, followed by size‑exclusion chromatography, to isolate the target compound. Verify purity using high‑performance liquid chromatography and mass spectrometry.
Store the final preparation at –80 °C in aliquots protected from light. Record all batch identifiers, culture conditions, and analytical results in a laboratory information management system for traceability and future reference.