Histological age estimation of the eggs of Robineau Desvoidy (Diptera: Calliphoridae).

Aging blow fly eggs can be critical to a forensic investigation, but there are currently no forensically useful timelines describing internal anatomical changes in embryological development. This is partly due to the lack of an economical, rapid and technically simple histological technique to allow mass production of slides for research and casework. We present a histological method that uses a slightly modified standard laboratory processing run with 1 h fixation in 10% formalin, 2 h softening in Molliflex and Haemotoxylin and Eosin (H&E) staining. We also present a summary of the internal anatomical changes that can be visualized using our technique in the developing eggs of Robineau-Desvoidy (Diptera: Calliphoridae). We examined eggs from at least three different females grown at 15 °C and sampled at 6 h intervals, and eggs grown at 20 °C and sampled at 3 h intervals. Blind aging trials demonstrated that it is possible to accurately age material grown at 20 °C to within 6 h (but attempts to further narrow this interval resulted in errors in one-third of cases). It was also possible to see sufficient anatomical detail to age eggs preserved for forensic casework 5, 8, 9, 10 and 11 years previously. Additionally, we determined that section quality was improved by 5 s fixation in hot water prior to preservation in ethanol. However, hot water fixation for longer than this increased the level of section artefact.