A practical approach to the sampling, fixation, softening, and sectioning of whole honey bees for histologic evaluation

Abstract

The Western honey bee ( Apis mellifera) is economically important as the primary managed pollinator of many agricultural crops and for the production of various hive-related commodities. Honey bees are not classically or thoroughly covered in veterinary pathology training programs. Given their unique anatomic and biological differences from the other species more traditionally evaluated by veterinary pathologists, establishing routine and consistent methods for processing samples for histology ensures accurate diagnostic and research conclusions. We developed and tested several field protocols for the sampling of honey bees. We compared the tissue-quality outcomes for worker bees fixed, collected, and/or softened under the following protocols: 1) routine formalin fixation; 2) softening chitin via exposure to Nair for 2 d or 3) 5 d; 4) shortened times between formalin submersion and trimming of body segments to enhance penetration of formalin into internal tissues; 5) ethanol submersion of specimen prior to formalin fixation; 6) indirect dry ice exposure; and 7) prolonged −80°C storage. Routine formalin fixation, exposure to Nair for 2 d, indirect dry ice exposure, and trimming body segments within 2 h of formalin submersion resulted in the highest quality histologic tissue sections. The poorest quality sections resulted from softening of chitin by exposure to Nair for 5 d, submersion in ethanol for 3 d before formalin fixation, and prolonged storage at −80°C. Our results indicate that routine formalin fixation is adequate, and that immobilizing bees with indirect dry ice exposure aids in sample collection without negatively impacting the quality of histologic sections.