Prepared by

ROY ELLIS

IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011

Principle

Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are ß-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species (Lyon H 1991).

The leprosy bacillus is much less acid and alcohol fast than the tubercle bacillus, therefore alcohol is removed from the hydrating and dehydrating steps and 10% sulphuric acid is used as a decolouriser in place of acid / alcohol solution. The sections are also deparaffinised using peanut oil/xylene mixture, this helps to protect the more delicate waxy coat of the organisms.

Technical Points

(step 7) It is important not to over-stain with methylene blue, as it will not be possible to remove the excess dye in alcohol.

Method

1.   Warm sections and de-paraffinize in a mixture of two parts xylene/one part vegetable oil for 15 mins.

2.   Blot dry and wash in water. Repeat if any xylene-oil remain on the section.

3.   Filter on carbol fuchsin solution, DO NOT HEAT, for 20 mins.

4.   Wash in running tap water.

5.   Differentiate in 10.0% sulphuric acid for 2 mins.

6.   Wash well in running tap water, rinse distilled water.

7.   Counterstain in 0.25% methylene blue for 20 seconds.

8.   Wash and blot dry. DO NOT DEHYDRATE IN ALCOHOL.

9.   Clear in xylene. Repeat the blotting-xylene treatment until section is clear.

10. Mount in a DPX type mountant.

Results

• Leprosy bacilli , hair shafts ……………magenta
• Nuclei, background…………………………blue
• Red blood cells……………………………… pale pink                       

Reagent Formulae

1.      xylene/peanut oil

xylene --------------------- parts

peanut oil ------------------- 1 part

2.      carbol fuchsin                  obtain from MedVet          

3.      sulphuric acid 10%          *add acid to water*  

                                               distilled water --------------- 90.0 ml

                                               conc. sulphuric acid -------- 10.0  ml

4.   acetified methylene blue     methylene blue (CI 52015) ----- 0.25 gm

                                              distilled water ------------------- 99.0 ml

                                               glacial acetic acid --------------- 1.0 ml

Dissolve the dye in the water. Add the acid and mix. Filter into the reagent bottle. The stain keeps well.

References

Faraco 1938

Fite, G.L., Cambre, F.J. and Turner, M.H.  1947, Procedures for demonstrating lepra bacilli in paraffin sections. Archives of Pathology, V43,    p624-625