Sterilization of Chrysomya putoria (Insecta: Diptera: Calliphoridae) eggs for use in biotherapy.

Abstract

Large-scale, quality-controlled laboratory production of fly larvae is needed for biotherapy. The objective of this study was to assess the action of glutaraldehyde on the sterilization of Chrysomya putoria eggs by applying pharmaceutical sterility tests. Egg masses with 0.600 g were divided into three parts of 0.200 g, the eggs were separated using sterile distilled water, and the suspensions obtained were mixed with activated 2% glutaraldehyde solution. After 15-min contact, the suspensions were filtered through Whatman filter paper, and the glutaraldehyde residue obtained in the filtrate was neutralized by rinsing with Tryptone Soy Broth. The treated eggs were placed aseptically on Petri dishes containing gauze moistened with sterile saline solution. About 10% of the sterilized mass was transferred to test tubes containing Tryptone Soy Broth and Fluid Thioglycollate Broth. The tubes were incubated, respectively, at 22.5 and 35.0°C for 14 d to verify egg mass sterility. The plates containing the rest of the eggs (90%) were sealed with plastic film and kept in a climatized chamber at 30°C/d, 28°C per night, 60 ± 10% relative humidity, and under a 12-h light period to assess insect viability and survival. Each experiment was carried out in triplicate using a biological class II safety cabinet. No change in color or turgidity was observed with the agent tested, proving the sterility of the product and that there was no trace of contamination. Forty larvae (in three replications) in the periods of 12, 24, and 48 h after sterilization, when transferred to diet, produced larvae, pupae, and total viability similar to the control (larvae without sterilization). However, for the 72- h treatment, larvae and total viability were significantly lower than for the other treatments. There was no significant difference for the pupal stage. The product tested was shown to be efficacious for use as a sterilizer of C. putoria eggs for all the parameters assessed.