18.104.22.168. Transmission electron microscopy (TEM)
Several fixation procedures are available for studies of microsporidia infections. The following methodology has been widely used with good results for both N. apis (Fries, 1989; Fries et al., 1992) and N. ceranae (Fries et al, 1996, Fig. 4):
- Prefix tissue specimens for transmission electron microscopy using 4 %. glutaraldehyde (v/v) in 0.067 M cacodylate buffer, pH 7.4, for 3 days to three weeks
- Keep material refrigerated (+7 °C) during prefixation.
- Wash in cacodylate buffer.
- Post fix for 2 hours in 2 % OsO4 (w/v) in 0.1 M S-colloidine buffer.
- Dehydrate through ethanol series at room temperature:
5.1. 5 min. in 30 % EtOH,
5.2. 5 min. in 50 % EtOH,
5.3. 5 min. in 75 % EtOH,
5.4. 5 min. in 95 % EtOH,
5.5. 3 X 10 min. in 100 % EtOH,
5.6. 3 X 10 min in 100 % propylene oxide.
- Embed in epoxy resin (Agar 100) by routine procedures for electron
The number of polar filament coils is one tool that helps to differentiate between species of Nosema (Burges et al., 1974). In N. ceranae, the number of filament coils varies between 20 and 23 in mature spores (Fries et al., 1996), whereas the number of polar filament coils in spores of N. apis is always larger and often more than 30 (Fries, 1989). The immature spores, where the filament is still developing, can be distinguished from mature spores on the less developed spore wall.
Fig 4. Transmission electron micrograph of N. ceranae infected tissue: host nucleus (HN); healthy tissue (HT); infected cell (IC); dividing stages (DS); immature spore (IS). Bar= 10 mm. Photo: I Fries.