For the diagnosis and detection of Nosema spp.-infected colonies, the oldest honey bees should be the target population, since they are more frequently infected compared to younger bees (Meana et al., 2010; Smart and Sheppard; 2011).  Forager bees can be sampled outside the entrance. This method is useful in all areas during flight and foraging conditions. Caution should be taken to avoid collection of young bees performing their orientation flights. The time of the day of these flights could change in different geographic areas. Bees that conduct their orientation flights are easily recognized by the hovering behaviour in large numbers outside the entrance. When this behaviour is seen, attempts to sample for foragers should be avoided.

During non-flight conditions, old bees may still be the target population for diagnosis. To avoid sampling of newly emerged, uninfected bees, the samples can either be taken from peripheral combs in the brood area, without hatching bees, or in a super above a queen excluder. The variation introduced by not sampling flight bees is unfortunate, because it reduces the possibility for meaningful comparisons between sites. Nevertheless, when samples are taken during late autumn or winter or during the active season outside of foraging conditions (e.g. study of infection development across the year), bees from within the hive must be sampled.

To determine the degree of infection within colonies, both pooled sampling or individual sampling of bees can be used (see section Samples should preferably be stored in the deep freezer until further processing