Staining Methodification of Ziehl Neelsen Method,Leprae,Tuberculosis,Mycobacteria,Brucella,Loefflers Methylene Blue

Modifications of Ziehl-Neelsen Method

Modifications of Ziehl-Neelsen method
Ziehl-Neelsen method gives better result when you desire to stain all acid-fast bacteria. Following modifications are useful in special cases.

(i) 5% sulphuric acid is used as the decolourizing agent to stain M. leprae. They are acid-fast, but to a lesser extent than, M. tuberculosis.

(ii) 3% HCI in 95% ethanol is used as decolourizer to stain M. tuberculosis, since other organisms are acid-fast, but M. tuberculosis is both acid as well as alcohol fast.

(iii) To demonstrate acid-fast clubs caused by Actinomyces, Mycobacteria and Nocardia, 1 % sulphuric acid is used the decolourizer. Cultures of some Nocardia sps. show acid fastness when decolourized with 0.5% sulphuric acid

(iv) Brucella abortus in infected material can be demonstrated easily by staining the smear or sections without heat treatment for 15 minutes with 1:9 diluted carbol fuchsin solution (S19). This is followed by decolourization with 0.5% acetic
acid for 15-20 seconds. Wash and counterstain with Loefflers methylene blue (S₁₈).

Acid-fast organisms will be stained red, while other bacteria and background will appear green or blue according to the counterstain used. Note the arrangement of acid-fast organisms, e.g. chinese letter arrangement-M. tuberculosis and cigarette packet arrangement-M. leprae.

If malachite green is used as a counterstain, use of deep green filter in the light source may facilitate the observation. This renders the counterstain invisible and acid-fast bacteria black.

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Modifications of Ziehl-Neelsen Method