Hyphal tip isolation of A. apis strains

A single honey bee larva can ingest a large number of fungal spores, thus to ensure that the isolate is composed of a single strain, hyphal tip isolation is preferred (Fig. 4).

  1. Place a dissecting microscope in a sterile hood and wipe with 70 % ethanol
  2. Use a fresh A. apis isolate (approx. 4 cm in diameter)
  3. Find a single hypha at the edge of the culture under the microscope, and cut a hyphal tip just before the last branching point using a scalpel and a minutien pin
  4. Transfer the tip to a new culture plate
  5. Incubate the plate in darkness at 30-34 °C

Addition of antibacterial compounds (e.g. chloramphenicol, cycloheximide, streptomycin or dodine) in the media can be advantageous to prevent bacterial growth. Ascosphaera apis growth will often be inhibited, but once transferred back to non-antibiotic media it can be quickly recovered.

Fig. 4. Hyphal tip isolation of Ascosphaera apis.

Figure 4