1.3.1. Quantify the level of fungal infection in bee colonies

Different methods have been used to assess the level of chalkbrood infection in the honey bee hive, but none of the methods are completely reliable. The most direct method is to remove brood combs from the hives and count the number of mummies inside capped and uncapped cells. These values can be added to the number of mummies found on the bottom board of the hive. It is however important to add an entrance hive trap like a pollen trap (see the BEEBOOK paper on miscellaneous methods (Human et al., 2013) or a screened bottom board (Flores, unpublished data), to hinder the bees from removing mummies from the hive. 

Assessing chalkbrood infection by counting mummies has important drawbacks: it does not include the number of mummies inside capped cells, unless all capped cells are opened and inspected; and it does not include the mummies that are removed from the hive in small pieces by worker bees which are thus not collected by the entrance traps. Furthermore, these counting methods are heavily influenced by environmental conditions. High nectar flow induces higher removal of mummies (Thompson, 1964; Momot and Rothenbuhler, 1971) and during a nectar flow more mummies may be recovered in the traps, leading to possible error in the estimation of disease prevalence.

For the above reasons, we propose that the level of colony infection be evaluated by chilling the brood to 25ºC (Flores et al., 1996a). The number of mummified larvae after chilling allows for an assessment of the degree of potential infection in bee colonies. This method has also been used to compare the degree of infection among colonies before and after a treatment or as a routine colony inspection (Flores et al., 2001; 2004a). To minimize the effects of brood removal by workers, it is important to place the brood comb in an incubator, as described in paragraph Procedure for quantifying the level of colony infection