Preparation of dipteran larvae for scanning electron microscopy using a freeze-substitution technique

Abstract

Difficulties are often encountered in achieving good preservation of dipteran larvae for scanning electron microscopy. Standard fixation methods frequently fail because fixatives do not penetrate the cuticle rapidly, resulting in distortion and generation of artifacts on the surface. A freeze-substitution technique that produces excellent preservation of the larval stages of a wide variety of Diptera is described. The method employs Freon 12 for rapid freezing of the specimens and methanol as the substitution fluid. The technique is simple, inexpensive, and demonstrates a significant improvement in preservation of specimens.