Acid fast staining is used to stain bacteria with mycolic acid in their cell walls. The Ziehl-Neelsen staining method is used which involves mixing carbolfuchsin with phenol and heating the cells for 5 minutes. Phenol allows the penetration of carbolfuchsin into the cell wall and the heat further fixes the stain. The cells are then treated with acid-alcohol which acts as a decolorizer however it does not remove the stain from the mycolic acid-carbolfuchsin complex. These cells are referred to as acid-fast. Cells that do not contain mycolic acid in their cell walls are decolorized by the acid-alcohol and referred to as non-acid-fast. Non-acid-fast bacteria are counterstained with methylene blue and appear blue under the microscope. Acid-fast cells appear red to pink in stained preparations. Heating the cells with phenol can create fumes that may damage mucus membranes and eyes. The staining method may be modified to prevent these fumes. The concentrations of phenol and carbolfuchsin may be increased however no heat is used.
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