2.2.2.1. Light microscopy (LM)

A compund microscope using 400X magnification is sufficient for observing Nosema spp. spores in macerated bee preparations. Use of phase contrast light microscopy facilitates distinguishing spores of microsporidia from yeast or other particles.

Although the differences in spore size between N. ceranae and N. apis are not immediately apparent in light microscopy, there is a consistent difference. Spores of N. ceranae are clearly smaller compared to spores of N. apis. Fresh, unfixed spores of N. apis measure approximately 6 x 3 µm (Zander and Böttcher, 1984); whereas, fresh spores of N. ceranae measure approximately 4.7 x 2.7 µm (Fries et al., 1996) (Fig. 2). Although there is a slight overlap, with the smallest N. apis spores being smaller than the largest N. ceranae spores, the average spore size of N. apis is approximately 1 µm larger in length (Fig. 2).

In contrast to spores of N. apis, the spores of N. ceranae are often slightly bent, and appear less uniform in shape compared to N. apis spores (Fries et al., 1996; Fig. 2). Although the difference in the size of spores between these species is clear, it may still be difficult to detect the difference in routine diagnosis of infected bees using light microscopy. This is particularly true because mixed infections of both species can occur (Chen et al., 2009), even in individual bees (Burgher-MacLellan et al., 2010).

Because of their light refractive properties, spores of Nosema spp. are easily seen without contrast colouring in the light microscope in water squash preparations at 200-400 X magnifications. Methanol-fixed smears contrast coloured using Giemsa staining is the standard technique for microsporidians and is described in section 2.2.2.1.1. However, the nuclei are not revealed because of the staining of the spore contents. Giemsa staining can be useful to visualize infection in tissue preparations. Another method to identify Nosema species with LM is to mount sections of material embedded for electron microscopy (section 2.2.2.2) for light microscopy after contrast colouring with toluidine blue (section 2.2.2.1.2). For a further range of colouring techniques for microsporidians see Vavra and Larsson (1999).

Fig. 2. Spores of Nosema ceranae (A) and Nosema apis (B) in light microscopy squash preparations. Bars = 5 mm. From Fries et al., 2006.

Figure 2

2.2.2.1.2. Toluidine staining