Preservation, Labelling and Chain of Custody

Blow fly larvae have longitudinal muscles that contract sharply when exposed to a sudden chemical irritant such as ethanol. A larva dropped live into a vial of ethanol shortens by as much as 20-30% before it dies, depending on instar and species. Body length is one of the primary measurements used to assign a larva to its development stage on the species growth curve. A specimen 25% shorter than its true size will appear to be at an earlier developmental point, making it seem younger than it was, which in turn shifts the PMI estimate toward a more recent death.

The standard protocol addresses this directly. Bring water to approximately 80 degrees C, not boiling, because boiling water can damage delicate features used in identification. Immerse the larvae for 30-60 seconds. Remove and blot briefly on clean tissue, then transfer immediately to the ethanol vial. The heat kills via protein denaturation before any muscular contraction has time to occur. The resulting specimen holds its living length.

Eggs and puparia do not require hot-water killing. Eggs are fixed directly in ethanol. Puparia are already in a protective hardened case and are stored directly in ethanol. The hot-water step applies specifically to soft-bodied larvae.

After hot-water killing, specimens are transferred to 70-80% ethanol and should remain in ethanol for all subsequent storage. The concentration matters. Ethanol above 90-95% dehydrates larval tissue too aggressively, making the cuticle brittle and causing specimens to crack during later examination. Ethanol below 60% does not fix tissue adequately and allows enzymatic and microbial decay to continue slowly inside the specimen, degrading morphological characters over weeks to months.

For long-term storage beyond six months, the ethanol should be changed once after the first 24-48 hours. The initial fixation draws water and biological fluids out of the specimen, diluting the ethanol. Replacing this first fixative with fresh 70-80% ethanol maintains adequate preservation. Specimens stored without a change-over in diluted ethanol show tissue softening on dissection.

Every container, whether a killed-sample vial, a rearing container, a soil bag, or an adult-insect tube, receives a unique sequential sample number that links it to the scene notes and the chain-of-custody form. The label must be readable after extended storage in ethanol, which means pencil or archival-grade waterproof ink on paper labels, not ballpoint pen on adhesive labels applied to the outside of glass vials.

• External label on vial or container: case number, unique sample number, date and time, collection location, instar if known, fixative, collector's initials.
• Internal label inside vial (for ethanol vials): a small piece of acid-free paper written in pencil, rolled and inserted before capping. This is the backup if the external label is damaged or falls off during storage.
• Rearing container label: same fields, plus the rearing food source, rearing temperature, and start date. Updated with emergence date and adult identification when rearing is complete.
• Soil bag label: collection depth, body zone, collection date and time, any associated insects in the soil sample.

Standard chain-of-custody forms used for physical exhibits track who held the item and when. For entomological samples, this framework requires additional fields because the sample is biologically active at collection and can change state during handling. A rearing container that spent eight hours at ambient temperature in transit is not the same biological sample as one that spent eight hours at 4 degrees C; both are physically intact, but the developmental clock ran at different rates.

The entomology chain-of-custody record should capture, for each sample: the method and conditions of collection, the killing protocol used (or that the sample is live-rearing), the fixative and concentration, the temperature during transport, the time between collection and laboratory receipt, and each transfer with a date, time, and two signatures. For rearing samples, the record continues with the rearing log until adult emergence.

1. Collection entry
   Date, time, scene location, body zone, method (forceps, aspirator, net), killing protocol, fixative, ambient temperature, maggot mass temperature, collector name and signature.
2. Scene-to-mortuary or laboratory transfer
   Transport container type, temperature maintained during transport, departure and arrival times, name and signature of person transporting, name and signature of receiving person.
3. Laboratory receipt
   Date and time received, condition on arrival, storage location assigned, receiving scientist name and signature. Killed samples enter storage; rearing samples enter the rearing log.
4. Analysis and sub-sampling
   Any sub-sample taken for DNA, dissection, or comparison is recorded with the parent sample number, sub-sample number, purpose, analyst name, and date.

The five most common preservation and documentation errors in forensic entomology each undermine a distinct part of the evidential chain.

1. Live larvae into cold ethanol. Causes contraction and shortening. Instar may be assigned incorrectly; PMI estimate shifted younger. No recovery once fixed.
2. Too-dilute ethanol. Decay continues inside the specimen. Morphological characters used for instar and species identification degrade over weeks. Problem may not be visible until dissection months later.
3. Sealed rearing container without ventilation. CO2 accumulates; larvae die within hours. Rearing sample lost; species identification by adult emergence is no longer possible.
4. Ethanol-soluble ink on labels. Label ink dissolves into the ethanol over weeks, rendering the label blank. Without identification, the vial may need to be excluded from evidence.
5. Killed and rearing fractions in the same container. Even if initially correct, the killed specimens begin to decompose in the absence of fixative. The rearing larvae die from ethanol vapour if the containers are stored together unsealed. Separate physical containers, separately labelled.