7. Apply safranin stain or neutral red (secondary dye or counterstain) to the specimen

8. Wash the specimen and allow to air dry.

9. The specimen is ready to be viewed under the microscope. Gram-positive bacteria appear purple and gram-negative bacteria appear pink.

ZIEHL-NIELSEN STAINING TECHNIQUE

1. Prepare the specimen (make a Smear).

2. Apply the red dye carbol-fuchsin stain generously.

3. Let the specimen sit for a few minutes.

4. Warm the specimen over steaming water. The heat will cause the stain to penetrate the cell wall.

5. Wash the specimen with an alcohol-acetone decolorizing solution consisting of 3 per cent hydrochloric acid and 95 per cent ethanol. The hydrochloric acid will remove the colour from non–acid-fast cells and the background. Acid-fast cells will stay red because the acid cannot penetrate the cell wall.

6. Apply methylene blue stain (secondary dye or counter stain) on the specimen.

7. Observe under the microscope. Acid fast bacilli will appear pink while the background appears blue.