Safety

• Cryogen: LN₂ can cause severe cold burns and asphyxiation. Use cryo‑gloves, lab coat, closed shoes, and a face shield; work in a ventilated area; keep dewars upright.
• Biological: Treat specimens as potentially biohazardous (agricultural pest). Wear gloves; avoid cross‑contamination.
• Sharps & fieldwork: Use forceps/scissors carefully; follow local field safety procedures.
• Never seal LN₂ inside a container. Use cryo‑rated vials and safe freezing practices (see Procedure).

Materials & Equipment

• Field collection tools: forceps/soft brushes, scissors, vials, collection jars.
• Labels/marker (solvent‑resistant) or barcoded cryo‑labels.
• Cryo‑rated external‑thread cryovials (1.5–2 mL) or pre‑cut aluminum foil “boats.”
• Liquid nitrogen in approved dewar or dry shipper; cryo‑racks/floats; metal tongs.
• Insulated transport container for frozen samples (dry shipper or dry‑ice cooler).
• −80 °C freezer and −20 °C freezer (staging only if needed).
• PPE: cryo‑gloves, nitrile gloves, lab coat, face shield/safety glasses.
• Optional: RNase/DNase‑free wipes, 70% ethanol for tool cleaning, GPS/phone for coordinates, field notebook or ELN.

Definitions

• Snap‑freeze: Rapidly freezing tissue by direct exposure to LN₂ (liquid or vapor phase) to minimize molecular degradation.
• Whole tissue: Entire organism or intact body part without prior homogenization.
• Cold anesthesia: Temporary immobilization on ice; preferred over chemical anesthetics for molecular work.

Procedure A — Field Collection

1. Identify and collect target life stage(s) with minimal handling stress. For larvae on host plants, gently remove with soft forceps or a brush.
2. Record metadata immediately: date/time (local), GPS coordinates, habitat/host plant, life stage, sex (if known), treatment/exposure, temperature/weather.
3. Temporary holding (brief): Keep specimens cool and shaded; avoid water in containers. Aim to freeze within minutes of capture when molecular preservation is critical.

Procedure B — Snap‑Freezing Whole Tissue with LN₂

Choose one of the two safe methods:

Method 1 — Foil boat snap‑freeze (preferred to avoid LN₂ ingress)

1. Place specimen in a pre‑labeled aluminum foil boat held with tongs.
2. Immerse the boat into LN₂ (liquid phase) for ≥30–60 s (larger stages may require 2–3 min) until fully rigid and frosted.
3. Transfer the still‑frozen specimen into its matching pre‑labeled, pre‑chilled cryovial. Cap tightly.

Method 2 — Vial rack snap‑freeze (cryo‑vials only)

1. Place the specimen directly into a cryo‑rated, external‑thread vial; cap tightly.
2. Position the vial in a cryo‑rack and lower into LN₂ (liquid or vapor phase) for ≥2–3 min until fully frozen.
3. Keep caps above the liquid when possible; ensure no LN₂ enters the vial. Do not use non‑cryo tubes.

For multiple specimens: Freeze one individual per vial to avoid cross‑contamination and enable per‑sample metadata.

Procedure C — Transfer to −80 °C Storage

1. After snap‑freezing, place vials immediately into a pre‑chilled cryobox.
2. Maintain cold chain (LN₂ dry shipper or dry ice) during transport to the lab.
3. Store samples at −80 °C in a mapped location (freezer, shelf, rack, box, position).
4. Avoid freeze–thaw cycles: Do not open vials until processing; if aliquots are needed later, plan multiple individuals or split tissues at first thaw.

Procedure D — Shipping (if applicable)

1. Ship on dry ice (UN1845) or in a charged dry shipper; include absorbent and secondary containment.
2. Label the package per carrier and regulatory requirements; include a packing list and contact details.
3. Record shipment tracking and receiving confirmation.

Quality Control & Acceptance Criteria

• Time‑to‑freeze: Preferably <5 min from capture to LN₂ for sensitive molecular endpoints.
• Freeze confirmation: Specimens appear rigid/opaque with no soft spots before −80 °C transfer.
• Label integrity: All vials legible and traceable to complete metadata.
• Temperature integrity: Dry shipper/dry‑ice levels adequate; −80 °C logs within set limits.
• Audit sample: Periodically thaw a non‑critical specimen to assess integrity (e.g., DNA QC on gel, RNA RIN, or housekeeping qPCR).

Troubleshooting

• Ice inside vials / popping on warm‑up: LN₂ ingress. Use foil‑boat method or ensure cryo‑rated vials and keep caps above liquid when possible.
• Poor downstream nucleic acids: Slow freezing or thawing events. Shorten time‑to‑freeze; verify cold chain; consider processing on dry ice during any handling.
• Label loss: Use cryo‑labels/solvent‑resistant marker; add an internal paper tag with ID.
• Specimens stick to tools: Pre‑cool tools; use a minimal dusting of sterile foil to separate.
• RNase/DNase concerns: Clean tools with 70% ethanol and RNase/DNase decontaminant between samples; change gloves frequently.

Documentation (ELN or Field Sheets)

Record for each specimen:

• Unique Sample ID; collector; date/time; GPS coordinates; site description/host plant.
• Life stage; sex (if known); treatment/exposure; ambient temperature/weather.
• Freezing method (foil vs vial rack), time‑to‑freeze, LN₂ dewar ID, box/rack/freezer location.
• Chain‑of‑custody (handlers, transfers); shipping details if any.
• Deviations, incidents, or QC notes.

Storage & Retention

• Primary storage at −80 °C; recommended use within 12–24 months for RNA‑centric work, longer for DNA if temperature logs are in range.
• For very long‑term archiving, consider LN₂ vapor phase storage (if available) with appropriate inventory control.

Waste & Decontamination

• Return unused LN₂ to dewar; do not cap LN₂ in sealed containers.
• Dispose of plant debris/unused specimens per biosecurity rules.
• Clean tools and work surfaces (70% ethanol; RNase/DNase remover if needed).