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.
Step 3: Place a piece of paper towel on top of the dye. Be sure the paper towel is saturated with the dye.
Step 4: Dry heat for 2 minutes.
Step 5: Cool and rinse with water.
Step 6: Decolorize with acid-alcohol for 15-20 seconds.
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.
Step 9: Wash and blot the slide with bibulous paper.
Step 10: Focus 10X - then use oil immersion.
| 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.
