Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as Nocardia.
Preparation of microscope slide:
- Clean slide with a Kimwipe and alcohol to remove any fingerprints.
- Draw two circles with your Sharpie on the bottom of the slide.
- Using your inoculation loop, put two small drops of water in each circle.
- Using aseptic technique, remove a very small amount of bacteria from the culture tube. Make sure you flame the tube before and after you enter.
- Smear the bacteria in the drop of water on your slide. You may go out of the perimeter of your circles!
- Let the slide air dry completely.
- Heat-fix the slide by running it through the flame 3-4 times with the ‘smear’ side up. Do not flame the side with the bacteria!
- Let the slide cool completely and you are ready to stain it.
- Cover the smears with a piece of paper towel within the border of the slide.
- Place the slide over a beaker of steaming water. Do not let the beaker boil dry
- Flood the paper towel with carbolfuchsin and let the slide steam for 3-5 minutes.
- Complete the rest of the procedure at the back sinks.
- Remove the stained paper towel and discard it in the trash can, not in the sinks.
- Gently rinse the slide with water to remove any pieces of a loose paper towel and tap dry.
- Apply acid-alcohol for 15-30 seconds.
- Rinse off and tap dry.
- Counterstain with methylene blue for 1.5 minutes.
- Rinse with water and tap dry.
- Blot gently with bibulous paper.
- Dry the bottom of the slide before placing it on the stage of the microscope and view with the oil immersion lens.