I believe the correct answer is add secondary stain
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Explanation:</h2>
The Ziehl-Neelsen stain (ZN stain), also called the hot method of AFB staining, is a type of differential bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Acid fast organisms are those which are capable of retaining the primary stain when treated with an acid (fast=holding capacity). Members of the Actinomycetes, genus Nocardia (N. brasiliensis and N. asteroides are opportunistic pathogens) are partially acid-fast.
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Further Explanation:</h2>
Make a thin smear of the material for study and heat fix by passing the slide 3-4 times through the flame of a Bunsen burner or use a slide warmer at 65-75 C. Do not overheat.
Place the slide on staining rack and pour carbol fuschin over smear and heat gently underside of the slide by passing a flame under the rack until fumes appear (without boiling!). Do not overheat and allow it to stand for 5 minutes.
Rinse smears with water until no color appears in the effluent.
Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide appears light pink in color (15-20 sec).
Wash well with clean water.
Cover the smear with methylene blue or malachite green stain for 1–2 minutes.
Wash off the stain with clean water.
Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).
Examine the smear microscopically, using the 100x oil immersion objective.
The phenolic compound carbol fuchsin is used as the primary stain because it is lipid soluble and penetrates the waxy cell wall. Staining by carbol fuchsin is further enhanced by steam heating the preparation to melt the wax and allow the stain to move into the cell. Acid is used to decolorize nonacid-fast cells; acid-fast cells resist this decolorization. The ability of the bacteria to resist decolorization with acid confers acid -fastness to the bacterium. Following decolorization, the smear is counterstained with malachite green or methylene blue which stains the background material, providing a contrast colour against which the red AFB can be seen.
Acid alcohol can also be used as decolorizing solution, resistant organisms are referred to as Acid Fast Bacilli (AFB) or Acid Alcohol Fast Bacilli (AAFB).
Level: College
Subect: Microbiology
Topic: Bacteriology
