7.1: Acid-Fast Stain
- Understand the principle behind acid-fast staining and its application in the identification of Mycobacterium species.
- Demonstrate proficiency in preparing bacterial smears, heat-fixing slides, and staining with acid-fast stains.
- Interpret staining results accurately, distinguishing acid-fast bacteria from non-acid-fast bacteria.
- Differentiate the cell wall structures of acid-fast and non-acid-fast bacteria
- Describe the significance of acid-fast staining in the diagnosis of tuberculosis.
Acid Fast Stain
The Acid-fast stain is a differential stain used to identify acid-fast bacteria, particularly species of the genus Mycobacterium . Acid-fast bacteria are characterized by wax-like, nearly impermeable cell walls containing mycolic acid . This type of cell wall is resistant to most compounds, therefore acid-fast bacteria require a special staining technique.
The procedure involves primary staining with carbol fuchsin, decolorization with an acid-alcohol solution, and counterstaining with methylene blue. Carbol fuchsin is lipid-soluble and contains phenol, which helps the stain penetrate the waxy cell wall. Penetration is assisted by the addition of heat in the form of steam. Steam helps to loosen the waxy layer and promote entry of the primary stain. The smear is then rinsed with acid-alcohol and counterstained with methylene blue. Acid-fast bacteria, characterized by thick, waxy cell walls rich in mycolic acids, retain the primary stain despite decolorization, appearing as bright red rods under the microscope. Non-acid-fast bacteria, after being stripped of carbol fuchsin during decolorization, will stain blue with the counterstain (i.e., methylene blue). (Fig.1 & 3)
Acid-fastness is an uncommon characteristic shared by the genera Mycobacterium and Nocardia (weakly acid-fast). Because of this feature, this stain is extremely helpful in the identification of diseases caused by acid-fast bacteria, particularly tuberculosis and leprosy.
Mycobacterium's waxy coat contributes to antibiotic resistance, prevents desiccation (i.e., drying out), and provides protection against phagocytic cells.
Structure and Composition of the Acid-Fast Cell Wall
The mycobacterial cell wall resembles both gram-positive and gram-negative cells by having a peptidoglycan layer nearly as thick as gram-positive cell walls and an outer, waxy layer, mimicking the outer cell membrane of gram-negative bacteria. Mycolic acids compose approximately 60% of the acid-fast cell wall in the genus Mycobacterium .
The following is a list of the layers of the acid-fast cell wall beginning with the innermost layer (nearest to the plasma membrane):
- Layer 1: Layer of peptidoglycan
- Layer 2: Layer of arabinogalactan (a long carbohydrate composed of the sugars arabinose and galactose)
- Layer 3: Outer cell membrane containing mycolic acids
- Layer 4: The outer surface is studded with surface proteins that differ with the strain and species of the bacterium
Like the outer membrane of the Gram-negative cell envelope, porins are required to transport small hydrophilic molecules through the outer membrane of the acid-fast cell wall.
Mycobacterium tuberculosis
In hospital settings, the acid-fast stain is widely used as an initial, rapid screening tool for tuberculosis (TB), particularly when patients present with symptoms such as persistent cough, fever, and weight loss. Sputum samples from these patients are stained using the acid-fast technique to detect Mycobacterium tuberculosis (Fig. 4). Since the acid-fast stain can be completed relatively quickly, it provides a preliminary diagnosis that informs immediate patient care and infection control protocols.
In addition to the acid-fast stain, other diagnostic methods can offer higher sensitivity or specificity for TB. The Mantoux tuberculin skin test (TST) is frequently used to screen for latent TB infection, particularly in healthcare settings. This test involves injecting a small amount of purified protein derivative (PPD) under the skin and measuring the reaction after 48–72 hours. A raised, hardened area suggests exposure to TB, though it does not distinguish between latent and active TB. Other diagnostic methods are more suited for active TB. Culture techniques, the gold standard for TB diagnosis, allow for the growth and antibiotic susceptibility testing of live bacteria, though they take weeks to yield results. Molecular methods, such as polymerase chain reaction (PCR), can detect Mycobacterium tuberculosis DNA within hours and identify rifampicin resistance. Additionally, blood tests like interferon-gamma release assays (IGRAs) measure immune responses, providing a diagnostic tool for latent TB. Each of these methods complements the acid-fast stain, enabling more accurate diagnosis, confirming infection, and supporting targeted treatment in hospital settings.
Figure 4: Ziehl-Neelsen staining has rendered these Mycobacterium tuberculosis cells red and the surrounding growth indicator medium blue. (credit: modification of work by American Society for Microbiology)
Lab Instructions
- Label a slide with a wax pencil.
- Prepare a slide using either Staphylococcus epidermidis or Mycobacterium smegmatis (see simple staining lab to review smear preparation).
- Allow the slide(s) to air dry.
- Once the liquid has completely evaporated, heat fix the bacteria by passing it through your flame three times. If using a hotplate, place the slide in the center of the hotplate for 30 seconds.
- Take your heat-fixed slide to the fume hood (the fumes from carbol fuchsin can be toxic).
- Once the water is boiling, place your slide on the slide rack above the boiling water.
- Cover the area of your smear with a square piece of bibulous paper.
- Carefully apply the carbol fuchsin stain to the bibulous paper.
- Steam with the stain on the slide for 5 minutes while continuously applying more stain so the bibulous paper never dries out.
- Gently remove the paper with forceps and discard it in the small waste paper cup located under the fume hood.
- Put the slide on your staining rack and gently rinse with water.
- Decolorize with acid-alcohol (one drop at a time until the runoff is clear), then rinse with water.
- Counterstain with methylene blue for 2 minutes.
- Rinse with water and blot dry with bibulous paper.
- Examine under the 100x objective lens with oil immersion and record your results.
Attributions
- Acid Fast Stain by Jackie Reynolds
- Cell wall peptidoglycan in Mycobacterium tuberculosis : An Achilles’ heel for the TB-causing pathogen by Arundhati Maitra et al. is licensed under CC BY 4.0
- General Microbiology Lab Manual (Pakpour & Horgan) by Nazzy Pakpour & Sharon Horgan is licensed under CC BY-SA 4.0
- The Acid Fast Cell Wall by Gary Kaiser is licensed under CC BY 4.0
- Staining Microscopic Specimens by Nina Parker, Mark Schneegurt, Anh-Hue Thi Tu, Philip Lister, Brian M. Forster is licensed under Creative Commons Attribution License
Citations
- Becker K, Sander P. Mycobacterium tuberculosis lipoproteins in virulence and immunity–fighting with a double‐edged sword. FEBS Lett. 2016;590:3800–19.
- Centers for Disease Control and Prevention. (2023). Tuberculosis (TB): Clinical overview for healthcare professionals. U.S. Department of Health and Human Services. https://www.cdc.gov/tb/hcp/clinical-overview/index.html
- Forrellad MA, Klepp LI, Gioffré A et al.,Virulence factors of the Mycobacterium tuberculosis complex. Virulence . 2013;4:3–66.