THE VIRTUAL EDGE: Lab 3 Bacterial Staining Techniques II

Acidfast Stain: Background and Introduction

Mycobacterium and many Nocardia species are called acid-fast because during an acid-fast staining procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent acid-alcohol. Nearly all other genera of bacteria are nonacid-fast. The acid-fast genera have lipoidal mycolic acid in their cell walls. It is assumed that mycolic acid prevents acid-alcohol from decolorizing protoplasm. The acid-fast stain is a differential stain.

Ziehl Neelsen Acid-fast stain

ACID-FAST STAIN Cell Color Cell Color
Procedure Reagent Acid-fast Bacteria Nonacid-fast Bacteria
Primary dye Carbolfuchsin RED RED
Decolorizer Acid-alcohol RED COLORLESS
Counterstain Methylene blue RED BLUE


Step 2: Smear Preparation (Review)

  1. Add one loopful of sterile water to a microscope slide.
  2. Make a heavy smear of Mycobacterium smegmatis. Mix thoroughly with your loop. Then transfer a small amount of Staphylococcus epidermidis to the same drop of water.
    You will now have a mixture of M. smegmatis and S. epidermidis.
  3. Air dry and heat fix.

Step 3:

Cover the smear with carbolfuchsin dye. Carbolfuchsin a potential carcinogen. Please wear gloves and work with the stain in the hood.

carbolfuchsin dye

Place a piece of paper towel on top of the dye. Be sure the paper towel is saturated with the dye.

carbolfuchsin dye papertowel

Step 4:

Dry heat for 2 minutes.

Dry Heat

Step 5:

Cool and rinse with water.

Cool and rinse

Decolorize with acid-alcohol for 15-20 seconds.


Step 6:

Wash the top and bottom of slide with water and clean the slide bottom well.

Wash Slide

Step 7:

Counterstain with Methylene Blue for 30 seconds to 1 minute.

Counterstain with Methylene Blue

Wash and blot the slide with bibulous paper.

Wash and blot

Focus 10X - then use oil immersion. (Oil immersion review)


Lab 3 / Gram Stain / Acid Fast Stain / Lab 3 Organisms

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