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Some acid-fast microorganisms such as Tubercle bacillus and Mycobacterium leprae is unable to be stained because of the lipoidal material in the cell wall. But by the application of carbol fuchsin, dye molecular penetrates through lipoidal wall into cytoplasm. This process makes all cell appearing red and it is not easy to discolor. Based on this characteristic, decolorize samples with decolorizing reagent and counterstain with hematoxylin followed by dehydrate. In this process, the acid fast cells are resistant to decolorize and retain the first staining color (red), while non-acid fast cells lack the lipoidal material in the cell wall due to which they are easily decolorized, leaving the cells colorless. Then the decolorized cells absorb the counterstain and appear blue.
Sulfuric acid solution
Valid in 3 months at room temperature.
1、Deparaffinize and rehydrate to distilled water.
2、Immerse slides in Acid-fast staining solution for 30min, wash in running tap water.
3、Rinse in 1% hydrochloric acid to differentiate for 1 s, wash in running tap water for 2 min. Observe the presence of positive purple-red microorganism under microscopy.
4、Differentiate in Sulfuric acid solution for 1 s, wash in tap water. Repeat the operations until positive microorganism show purple-red in colorless background.
5、Stain with hematoxylin solution for 1-3 min, differentiate in 1% hydrochloric acid, wash in tap water, then back to blue in ammonia solution. Wash in running tap water.
6、Dehydrate in 3 changes of absolute alcohol, 5 min each. Clear in 2 changes of xylene, 5 min each. Then mount with resin mounting medium.
7、Microscopy determination and collect images for analysis.
Acid-fast microorganisms are purple-red and cell nucleus are blue.
1、Strictly control the extent in the two steps of differentiate. Over-differentiate may result in decolorization of acid-fast microorganism, while inadequate differentiate results in red background.
2、Hematoxylin counterstain should be controlled lightly so as not to affect observing positive results.