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Saturday, July 25, 2020

Acid fast staining procedure / acid fast staining principle


The technique was developed by paul Ehrlich (1832) and was modified later by Ziehl- Neelsen and therefore also known as Ziehl-Neelsen staining. This is a differential staining used to identify mainly the members of Mycobacterium especially Mycobacteriumtuberculosis (causing tuberculosis) and Mycobacterium leprae (causing leprosy). These organisms are difficult to satin by ordinary staining methods due to presence of high lipid content (mycolic acid) in their cell wall. Bacteria are classified as: 

what is acid fast staining and non-acid fast staining?

(I)  Acid fast if they retain the primary stain (carbol fuchsin) after the application of strong acid and appear red. 

(ii) Non-acid fast: If they do not retain the primary stain and are counterstained by methylene blue


Reagents:  i) Carbol fuchsin solution. 

(ii) Acid-alcohol solution: (3% Hel in alcohol) alternatively: Sulphuric acid solution (20-25%, v/v in water). 

(iii) Methylene blue counter stain (0.3%, w/v, aqueous) 

 Procedure of Acid fast staining : 

 (1) Prepare a smear of a purulent portion of the specimen on a clean glass slide. 

(2) Air dry and heat fix the smear. 

(3) Flood the smear with freshly filtered carbol fuchsin. Heat gently until steam rises. Continue to heat for 5 minutes so that steam is seen but without boiling. Do not allow the side to dry and add more stain from time to time to prevent this drying. 

(4) Cool and wash the stain off the slide with water. 

(5) Cover the slide with acid alcohol solution for 3 minutes. Wash with running tap water and drain. Repeat decolorization process until smear becomes faint pink in color. Finally wash with running tap water. 

(6) Cover the slide with methylene blue stain and leave it for 2 minutes. 

(7) Wash with tap water, blot dry or air dry the slide and observe under oil immersion objective 

Results: Acid fast organisms will appear bright red on a blue background while as non- acid fast organism will appear dark blue in color.

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