2.1.3.1.1. Cold

Freezing is a common method for killing adult honey bees because it can be easily and effectively applied, and will preserve genetic material. Freezing can, however, result in damage to cell structures, and therefore it is not recommended for studies that require internal tissues to remain intact, such as for quantifying hypopharyngeal development or midgut parasitism by Nosema spp. Exposing individuals to temperatures below -20°C will result in quick death; however, time required will vary depending on temperature and the number of individuals being collectively frozen (i.e. a higher number of honey bees collectively together will take longer to kill because of clustering behaviour). Placing individuals in a -20°C freezer for 2 hours is usually sufficient. Conversely, honey bees can be placed in a box of dry ice (e.g. Naug and Gibbs, 2009) or immersed in liquid nitrogen (e.g. Zayed et al., 2005) for near instant termination.