Veterinary Epidemiology and Zoonosis

EXERCISE-12: DIAGNOSIS OF ANTHRAX

Diagnois of anthrax is must in suspected cases of anthrax both in animals and human beings. The following tests can be performed to detect antigen in suspected cases of anthrax

• Polychrome methylene blue staining
• Ascoli's thermoprecipitation test
• Animal inoculation technique

Polychrome methylene blue staining (M’Fadyean reaction)

• Microscopic examination for anthrax bacilli to establish a ‘suspect’ diagnosis of anthrax and of environmental materials for the presence of anthrax spores is important to prevent spread of anthrax to humans.
• This is the ideal method for demonstration of the anthrax capsule. The capsule is stained pink and bacilli stained blue.
• If the carcass suspected for anthrax, blood smear from ear (dependant ear) vein or limb vein should be examined by staining technique.
• In positive cases, the carcass should not be opened. The carcass should be buried with lime powder or incinerated.

Preparation of polychrome methylene blue stain

• Methylene blue: 0.3 g
• Ethyl alcohol (95%): 30 ml
• Potassium hydroxide: 0.01 g
• Distilled water: 100 ml

This preparation is allowed to ripen for one year. The stain is kept in bottle, which is half filled and shaken at intervals to aerate thoroughly. The oxidation of methylene blue forms a violet compound that gives the stain its polychromic property. It may also be ripened quickly by the addition of 1 % potassium carbonate to the stain solution. 

Materials required

Materials required	Quantity and purpose
Microscopic slide	2 numbers
Absolute alcohol	Adequate quantity to fix the smear
Polychrome methylene blue stain	2 mL
Light microscope, scissor, forceps, tissue paper	Adequate numbers
Immersion oil	To examine the slide at 100X

Procedure

• Make two thin blood smears (from ear vein) on a microscope slide.
• Air dry and fix by dipping the slide in absolute alcohol for 30–60 seconds.
• Slide should not be heat dried to avoid distortion of morphology of the capsule.
• Flood with polychrome methylene blue stain.
• Wait for 30–60 seconds.
• Wash off the stain with tap water and air dry.
• Examine under oil immersion.

Interpretation of result

• Irregular pink capsular material surrounding the blue stained bacilli is indicative of anthrax organisms.

Note

• In clinical material, B. anthracis are Gram positive, thick, short, straight bacilli with square or truncated ends with parallel sides found usually single, in pairs or chains of three or four bacilli.
• The chain of bacilli with truncated and swollen ends gives a characteristic "bamboo stick" appearance.