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).

  1. 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.
  2. The contents then are passed through a fine strainer over a sink and rinsed with cold water to remove dissolved matter.
  3. 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.
  4. 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.
  5. 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).
  6. 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.
  7. 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.