How to prepare a tick for analysis? - briefly
Collect the tick with fine‑tipped forceps, place it in a sterile microcentrifuge tube, and keep it chilled (on ice or at 4 °C) until processing. Before molecular work, surface‑sterilize with 70 % ethanol, air‑dry, then freeze at –80 °C or store in RNAlater for downstream analysis.
How to prepare a tick for analysis? - in detail
Collect specimens with fine tweezers, grasping the tick close to the mouthparts to avoid damaging the body. Place each individual into a labeled microcentrifuge tube containing 70 % ethanol; label includes date, location, host, and collection code. Store tubes at 4 °C for short‑term or at –20 °C for long‑term preservation.
Prior to molecular work, remove excess ethanol by pipetting or brief air‑drying under a laminar flow hood. Surface‑sterilize specimens to eliminate external contaminants: immerse in 10 % bleach for 1 minute, rinse twice in sterile distilled water, then in 70 % ethanol for 30 seconds. Allow tubes to air‑dry before proceeding.
For morphological examination, mount the tick on a glass slide with a drop of glycerol or Hoyer’s medium. Position the specimen ventral side up, cover with a coverslip, and examine under a stereomicroscope. Record measurements (idiosoma length, scutum width) and photograph key structures.
DNA extraction follows a standardized protocol: place the tick in a 1.5 ml tube, add 180 µl of lysis buffer (e.g., ATL) and 20 µl proteinase K. Incubate at 56 °C for 2–3 hours, then proceed with silica‑column purification according to the manufacturer’s instructions. Elute DNA in 50 µl TE buffer and store at –20 °C.
For RNA analysis, use RNAlater or immediate flash‑freezing in liquid nitrogen, then store at –80 °C. Extract RNA with a phenol‑chloroform method or commercial kit, ensuring DNase treatment to remove genomic DNA.
When preparing samples for pathogen detection, set up PCR reactions with appropriate primers for target organisms (e.g., Borrelia, Anaplasma). Include negative extraction controls and positive reference DNA. Run amplification, verify products by agarose gel electrophoresis, and sequence amplicons if necessary.
Maintain a detailed laboratory notebook: record each step, reagent lot numbers, incubation times, and any deviations from the protocol. This documentation supports reproducibility and facilitates data verification.