6.1. Carbol fuchsin staining

Prepare the carbol fuchsin stain by mixing the following 2 solutions.

Solution A: 0.2 g basic fuchsin and 10 ml 95 % ethanol.

Solution B: 5 g phenol and 90 ml distilled water.

Procedure:

  1. Select larvae and/or pupae showing signs of European foulbrood and place them on a microscope slide.
  2. Using a swab or stick, pulp the larvae together and spread over the slide pushing any excess off one end, to leave a thin smear.
    Allow the smears to dry before processing.
  3. Heat fix by flaming the slide over a burner a few times
  4. Flood with 0.2 % carbol fuchsin for 30 seconds.
  5. Wash off the stain and either air dry or gently blot dry before microscopic examination at 1000 times magnification.

A diagnosis of European foulbrood is made if examination revealed M. plutonius-like organisms. Organisms are considered to be M. plutonius if they are lanceolate cocci, approximately 0.5 x 1.0 µm. E. faecalis is very like M. plutonius in appearance and has frequently been confused as being the causative agent (Bailey and Gibbs, 1962; Hornitzky and Wilson, 1989) (Figs 5 a, b, c).

An alternative to the 0.2 % carbol fuchsin stain is the Gram stain, useful mainly when the Gram positive feature of M. plutonius needs to be confirmed.

Fig. 5. a) Early infection: only Melissococcus plutonius. Arrow indicates a mass of coccoid/lanceolate M. plutonius organisms. b) Infiltration of secondary invader Paenibacillus alvei. Arrow indicates one of the many vegetative P. alvei cells. c) Proliferation of P. alvei spores to the virtual exclusion of M. plutonius. Arrow indicates one of the many P. alvei spores. Photos: Michael Hornitzky.

a)

Figure 5a

b)

Figure 5b

c)

Figure 5c