Acid-fast stain

Stock cultures of

Mycobacterium nonchromogenicum & Staphylococcus epidermidis

The acid-fast stain is what kind of stain

differential stain used to identify bacteria with a unique cell wall

Bacteria with an acid-fast cell wall contain large amounts of

glycolipids and mycolic acids linked to the peptidoglycan layer

Bacteria with an acid-fast cell wall contain large amounts of glycolipids and mycolic acids linked to the peptidoglycan layer. This distinctive waxy structure makes the cell wall…

hydrophobic (water-fearing) and so impermeable to most staining procedures and chemicals in aqueous (water containing) solution.

clinical specimens are often treated with very basic NaOH solutions in order to lyse, and so destroy, any potential contaminants within an individual sample prior to culturing for

Mycobacterium

• can survive this treatment while other cells in the specimen cannot

• so if Mycobacterium is present in the specimen, you can obtain a pure Mycobaterium culture in this manner

The acid-fast cell wall is exclusive to the genera

Mycobacterium and Nocardia (and just a few other species not mentioned here

acid-fast stain an especially important technique because it can be used as an important test for pathogenic species of

Mycobacterium such as M. tuberculosis, the etiological agent of tuberculosis; M. leprae, the etiological agent of leprosy; and M. avium-intracellulare complex (MAC), an atypical Mycobacterium that often causes tuberculosis-like disease in AIDS patients.

The waxy cell wall acts as a barrier to …

most antibiotics, preventing them from reaching their target sites, effectively making the cells antibiotic resistant.

Due to the difficulty in staining bacteria with this waxy outer layer, a special staining technique is employed that requires intense heating to force the primary stain, a red basic dye called

carbol fuchsin, into the cell wall.

Presumably, the mechanism of the acid-fast stain is the result of the relative solubility

carbol fuchsin and the impermeability of the cell wall.

What is more soluble in the phenol than in water, and phenol dissolves more easily in lipids than in acid-alcohol

fuchsin

What dissolves more easily in lipids than in acid-alcohol

phenol

carbol fuchsin has a higher affinity for

lipids than for acid-alcohol and will remain in the cell wall when the smear is washed with the acid-alcohol decolorizing solution.

Cells lacking the outer layer of glycolipids will be…..

decolorized and need to be counterstained with methylene blue at the end of the procedure.

Acid-fast bacteria stain

red

Non-acid-fast bacteria stain

blue

This staining technique consists of the following steps:This staining technique consists of the following steps:

1. Apply primary stain (carbol fuchsin, a basic red dye that contains 5% phenol)

2. Apply decolorizing agent (acid:alcohol). The primary stain is washed out, or decolorized, from non-acid-fast bacteria, while acid-fast bacteria retain the stain.

3. Apply counterstain (another basic dye, methylene blue) to stain the decolorized non-acid-fast bacteria blue.

Acid-Fast Staining Using the Ziehls-Neelsen Method

1. Prepare a single smear containing a mixture of M. nonchromogenicum and S. epidermidis. Be sure to use aseptic technique when preparing the mixture so you do not contaminate the pure cultures. Allow the smear to air dry completely.

2. After heat fixing the slide, flood the smear with carbol fuchsin for 60 seconds.

3. With the carbol fucshin still on the slide, use a slide holder to hold the slide, pass the slide through the flame of the burner until steam rises from the carbol fuchsin or place the slide on a slide warmer for 2 to 3 minutes. DO NOT boil the stain on the slide or allow it to completely dry out while heating.

• Note: Make sure the carbol fuchsin is heated in a well-ventilated area or in a fume hood. Make sure you use a slide holder, and NEVER your fingers, to hold a hot slide.

4. Once you have finished steaming your slide, allow the slide to cool. If using a slide warmer, use a pair of forceps to remove the slide from the heating surface and allow it to cool.

5. Gently rinse the slide with using your water bottle and decolorize the smear with acid alcohol for 30 seconds; do not over-decolorize.

6. Rinse the smear using your water bottle and drain any excess water.

7. Counterstain with methylene blue for at least one minute, then rinse with water and blot with bibulous paper.

8. Examine the finished slide under the oil immersion lens with your microscope.

• Note: Acid-fast cells will retain the carbol fuchsin dye through the decolorizing step and stain "hot pink". Cells that are not acid-fast will be decolorized and stained blue to purple by the counterstain depending on how well the slide was decolorized with acid-alcohol.
  

9. Draw the results of the acid-fast stained bacteria you observed under the oil immersion lens in the space provided in the "Exercise 7: Lab Worksheet" page found in the Exercise 7: Acid Fast Stain folder in the menu. Use colored pens or pencils to indicate which cells are acid-fast positive or negative.

Note:

• remember not to confuse this stain with the Gram stain. Both stains are similar in that they are differential and stain cells based on cell wall components.

• The Gram stain result, however, is based on the amount of peptidoglycan in the cell wall,

• the acid-fast stain result is based on the presence of glycolipids and mycolic acids.

• Additionally, the Gram stain is the most commonly used stain, while acid-fast is used specifically when Mycobacteria are suspected to be present.

What stain is used specifically when Mycobacteria are suspected to be present?

acid-fast

What stain is based on

the amount of peptidoglycan in the cell wall, while the acid-fast stain result is based on the presence of glycolipids and mycolic acids.

Why can't acid-fast bacteria be stained using the Gram stain reagents?

Cell walls of Acid- fast bacteria are hydrophobic

Which of the following choices list the staining reagents for the Acid-fast stain in the correct order?

Carbol fuchsin, acid alcohol, methylen blue

The Acid-Fast Stain is a useful diagnostic stain for which of the following genera of bacteria?

Mycobacterium & Nocardia

Acid-fast bacteria will stain with carbol fuchsin even with out heating during staining

false

Which of the following diseases is caused by acid-fast bacteria?

Tuberculosis and Leprosy

Due to their waxy, hydrophobic cell wall, acid-fast bacteria are resistant to many antibiotics.

True