Lab 7 Acid Fast Stains

Arrange the steps of the acid-fast procedure in the correct order.

1. Place slide over heated beaker of water 2. Apply carbolfuchsin to smear 3. Remove slide from heat and rinse 4. Apply acid-alcohol to smear and rinse 5. Apply methylene blue to smear and rinse 6. Blot slide and view under microscope

What is the role of acid-alcohol in the acid-fast staining technique?

Acid-alcohol is the decolorizing agent. After rinsing excess carbolfuchsin from the smear, acid-alcohol is used to decolorize the smear. This removes carbolfuchsin from non-acid-fast cells.

While performing the acid-fast stain procedure, you heat your fixed bacteria with carbolfuchsin for 5 minutes, let the slide cool, and then rinse with water. What is your next step?

Add acid-alcohol. Acid-alcohol is the decolorizing agent in the acid-fast staining procedure. Acid-alcohol removes carbolfuchsin from non-acid-fast cells.

While performing the acid-fast stain procedure, you add methylene blue to your smear for the appropriate amount of time and then rinse with water. What is your next step?

Blot slide dry. Methylene blue is used as the counterstain in the acid-fast staining procedure. It's also the last reagent used in the procedure. So once you've rinsed off the excess methylene blue, you'll need to blot the slide dry. Once dry, the slide is ready for viewing under the microscope.

What is the purpose of carbolfuchsin in the acid-fast staining technique?

Carbolfuchsin is the primary stain. Carbolfuchsin is a lipid-soluble dye that can penetrate the waxy cell walls of acid-fast bacteria. This red dye is used as the primary stain in the acid-fast procedure.

Which of the following correctly describes the cellular morphology and appearance of Staphylococcus aureus after performing an acid-fast stain?

Clusters of blue cocci

Which of the following describes the cellular morphology and appearance of Mycobacterium smegmatis after performing an acid-fast stain?

Dark-red bacilli

You INCORRECTLY perform an acid-fast stain on a smear from a mixed culture of Mycobacterium smegmatis (acid-fast cells) and Staphylococcus aureus (non-acid-fast cells). Your mistake is that you forget to use acid-alcohol during the procedure. If you viewed this slide with a microscope, what would each cell type look like?

M. smegmatis cells would appear red. S. aureus cells would appear purple. Acid-alcohol is the decolorizing agent. If you forgot to decolorize, both cell types would retain the red primary stain, carbolfuchsin. The water-soluble methylene blue counterstain would not penetrate the cell walls of the acid-fast M. smegmatis, so these cells would remain red. In contrast, methylene blue would penetrate the non-acid-fast S. aureus. With S. aureus retaining both carbolfuchsin and methylene blue, these bacteria would appear purple.

You INCORRECTLY perform an acid-fast stain on a smear from a mixed culture of Mycobacterium smegmatis (acid-fast bacilli) and Staphylococcus aureus (non-acid-fast cocci). Your mistake is that you forget to use carbolfuchsin during the procedure. If you viewed this slide with a microscope, what would each cell type look like?

M. smegmatis(acid-fast) cells would be colorless. S. aureus (non-acid-fast) cells would be blue. Normally, carbolfuchsin stains acid-fast cells red, but the question indicates that this step was omitted. Subsequent use of methylene blue will color only the non-acid-fast cells because methylene blue cannot penetrate the waxy cell wall of the acid-fast cells. This leaves the acid-fast cells colorless, and the non-acid-fast cells blue.

You INCORRECTLY perform an acid-fast stain on a smear from a mixed culture of Mycobacterium smegmatis (acid-fast) cells and Staphylococcus aureus (non-acid-fast) cells. Your mistake is that you forget to use methylene blue during the procedure. If you viewed this slide with a microscope, what would each cell type look like?

M. smegmatis(acid-fast) cells would be red. S. aureus (non-acid-fast) cells would be colorless. Methylene blue is used as the counterstain in the acid-fast staining procedure. Forgetting to use methylene blue would leave the non-acid-fast cells colorless after the acid-alcohol decolorization step. In contrast, acid-fast cells are largely stained by the primary stain (carbolfuchsin), so the lack of methylene blue does not affect their appearance.

Why do acid-fast cells NOT appear blue following the addition of methylene blue?

Methylene blue does not penetrate acid-fast cell walls. The waxy mycolic acid layer of acid-fast cell walls is impervious to water-soluble dyes such as methylene blue and even to the intense acid-alcohol decolorizing agent. Acid-fast cells retain the carbolfuchsin primary stain and remain red at the end of the acid-fast staining procedure.

While performing the acid-fast stain procedure, you add acid-alcohol drop by drop until no more stain runs from the smear. What is your next step?

Rinse with distilled water. Rinsing with water is necessary after each reagent is used in the acid-fast procedure. The acid-alcohol decolorizing agent, if not washed off, will interfere with the subsequent methylene blue counterstain.

A smear from a mixed culture of bacilli and cocci is stained with the acid-fast procedure. This is an image of the stained smear at 1000X total magnification. Which of the bacteria, if any, are acid-fast?

The bacilli are acid-fast. The acid-fast staining procedure stains the acid-fast bacteria red, because those bacteria retain the carbolfuchsin primary stain. The bacilli in the image are red, so they're acid-fast. In contrast, blue cocci are non-acid-fast.

Why is acid-alcohol used as a decolorizing agent rather than ethyl alcohol?

The mycolic acids in the cell walls of acid-fast bacteria make them resistant to acid-alcohol.

The acid-fast stain has diagnostic value in identifying bacteria that contribute to which of the following infectious diseases?

Tuberculosis and leprosy

After methylene blue is applied as a counterstain, acid-fast bacteria will appear __________ while non-acid-fast bacteria will appear __________.

dark red; blue

Just after the decolorizer (acid-alcohol) is applied, acid-fast bacteria will appear __________ and non-acid-fast bacteria will appear __________.

dark red; colorless

Most bacteria are easily stained by aqueous stains. The lipoidal material that comprises mycobacterial cell walls, however, does not bind to aqueous stains, instead requiring a stain such as carbol fuchsin, which is composed of 5% __________.

phenol