3.3.6. Thoracic disc method (TDM)
TDM is a technique developed for detailed assessment of mite infestations. TDM involves cutting a thoracic disc that contains the prothoracic tracheae.
The bee is placed, dorsal side down and pinned in place and a razor blade or scalpel is used to cut off the head; then a thin transverse section is cut from the anterior face of the thorax, resulting in a 1 to 1.5 mm section, which includes the trachea (Fig. 12).
- The discs are then heated on a hot plate (approximately 60ºC for a minimum of 2 h) in 5-10% potassium hydroxide (KOH) to dissolve the surrounding tissues.
- The contents then are passed through a fine strainer over a sink and rinsed with cold water to remove dissolved matter.
- The samples are returned to a hot plate to digest further for another hour after adding fresh KOH, if the muscle tissue has not been completely cleared.
- When the thoracic discs become transparent in the middle, leaving only the sclerotized tergites around the outside, they are sieved and gently rinsed with cold tap water.
- The discs are returned to the Petri dish and suspended in distilled water containing a few drops of aqueous methylene blue (1%) (Peng and Nasr, 1985).
- Tracheae are then examined for tracheal mites under magnification (ca. 20-40×) using a dissecting microscope with lighting from below. Even a small number of mites can be detected through this method.
- Once cleared, the tracheae are then individually mounted on slides and examined under a microscope (Shimanuki and Cantwell, 1978; Delfinado-Baker, 1984; Shimanuki and Knox, 1991). A modified version of the thoracic disc method is used for detection of HBTM in New Zealand. Sampled bees are frozen for at least 24 h to facilitate cutting the thorax.
Fig. 12. Positioning a bee to cut thoracic disks. The head is first removed, then a thin section of the thorax is sliced. From Shimanuki and Knox, 1991.