Background & 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 acidin their cell walls. It is assumed that mycolic acid prevents acid-alcohol from decolorizing protoplasm. The acid-fast stain is a differential stain. 



Step 1: Smear Preparation  

a. Add one loopful of sterile water to a microscope slide. 

b. 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. 

c. Air dry and heat fix. 

Step 2: Cover the smear with carbolfuchsin dye. Carbolfuchsin a potential carcinogen. Please wear gloves and work with the stain in the hood. 

slide covered wit red stain



Step 3: Place a piece of paper towel on top of the dye. Be sure the paper towel is saturated with the dye. 

slide with red stain covered with paper towel

Step 4: Dry heat for 2 minutes.

slide with stain and paper towel on heating grate 

Step 5: Cool and rinse with water. 

slide with red stained smear

Step 6: Decolorize with acid-alcohol for 15-20 seconds.

slide rinsed with acid-alcohol

Step 7: Wash the top and bottom of slide with water and clean the slide bottom well. 

Step 8: Counterstain with Methylene Blue for 30 seconds to 1 minute. 

slide covered with blue stain

Step 9: Wash and blot the slide with bibulous paper. 

slide with light blue smear

Step 10: Focus 10X - then use oil immersion.

Ziehl Neelsen Acid-fast stain 

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



Mycobacterium smegmatis & Staphylococcus epidermidis 

Pink rods = Acid-fast 
Blue cocci = nonacid-fast 

The acid-fast Mycobacterium retains carbolfuchsin and stains hot pink. The Staphylococcus epidermidis is decolorized and the counterstain colors them blue. 

acid fast stain with red bacilli and blue cocci
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