Acid-Fast Staining

• Acid-fast staining


  Staining technique used to identify bacteria with high lipid and wax content in their cell walls that do not stain well with traditional bacterial stains
• Mycobacteria


  Genus composed of approximately 100 recognized and proposed species, including the causative agents of tuberculosis (TB) and leprosy
• Mycobacterium tuberculosis Complex


  • M. tuberculosis
  • M. bovis
  • M. bovis bacillus Calmette-Guérin (BCG)
  • M. africanum
  • M. caprae
  • M. canettii
  • M. microti
  • M. mungi
  • M. orygis
  • M. pinnipedi
• Nontuberculous Mycobacteria (Runyon Classification)

  • Photochromogens (e.g. M. kansasii, M. asiaticum, M. marinum)
  • Scotochromogens (e.g. M. scrofulaceum, M. szulgai, M. gordonae)
  • Nonphotochromogens (e.g. Mycobacterium avium complex, M. celatum, M. xenopI, M. ulcerans)
  • Rapid growers (e.g. M. abscessus, M. fortuitum, M. chelonae)

  
• Mycobacterium tuberculosis


  • Ghost cells (faint unstained, beaded gram positive bacilli)
  • Non-flagellated
  • Non-motile
  • Non-encapsulated
  • Non-sporeforming
  • Strict aerobes
  • Resist decolorization by an acid decolorizer (AFB)
  • Longest chain of mycolic acid (more than 60%; strong hydrophobic molecule)
  • Stained gram neutral or gram ghost in Gram stain
  • Aerobic, rod shaped, thin and slightly curved (0.2-0.6um in diameter, 1-4um length)
  • Slow grower (5-10% carbon dioxide)
  • pH 6.5 - 6.8
  • 1-10 um length
• Acid fastness


  Mycobacteria take up dye with increased staining time or application of heat but resist decolorization with acid-ethanol
• The CDC recommends Biosafety Level 2 practices, containment equipment, and facilities for preparing acid-fast smears and culture for nonaerosolizing manipulations
• Effective disinfectants for Mycobacterium spp.

  • Amphyl (Reckitt Benckiser North America, Wayne, NJ)
  • 0.05% to 0.5% sodium hypochlorite
  • Phenol-soap mixtures
• Specimens for mycobacterial infections

  • Respiratory samples (sputum, tracheal or bronchial aspirates, bronchial alveolar lavage)
  • Urine
  • Gastric aspirates
  • Tissue (biopsy) specimens
  • Cerebrospinal fluid (CSF)
  • Pleural and pericardial fluid
  • Blood
  • Fecal specimens
• Sputum specimen collection


  • Collect at least 1 teaspoonful (5-10ml) for DSSM
  • For Xpert MTB/RIF, sputum sample should not be less than one (1) ml
  • Early morning - best specimen as it represents the pulmonary secretions accumulated overnight
  • Yellowish, muco-purulent, cheesy material
  • Greater than 25 WBC's/LPO or 5 WBC's/OIO
  • Presence of alveolar macrophages (dust cells)
• Sputum specimen collection procedure

  1. Clean mouth by thoroughly rinsing with water

  2. Breathe deeply, hold breath for a second or two, and then exhale slowly. Repeat the entire sequence two (2) more times

  3. Cough strongly after inhaling deeply for the third time and try to bring up sputum from deep within the lungs

  4. Expectorate the sputum into a container with a well fitted cap
• Tests for diagnosis of TB

  • DSSM (Direct Sputum Smear Microscopy)
  • Chest X-ray
  • TB culture and Drug Susceptibility Test
  • Tuberculin Skin Test
  • Xpert MTB/RIF assay
• Chest X-ray


  • Used to complement bacteriologic testing in making a diagnosis
  • Low specificity and does not differentiate drug-susceptible from drug-resistant disease
• TB culture and Drug Susceptibility test

  • Gold standard for the diagnosis of tuberculosis
  • Highly specific and sensitive
  • Solid media: Ogawa or Lowenstein Jensen
  • Liquid media: MGIT - Mycobacteria Growth Indicator Tube
  • Routine diagnostic test for drug resistant TB cases
  • Used for TB prevalence survey, drug resistance surveillance and research
• Tuberculin Skin Test (TST)


  Basic screening tool for TB infection among children using Purified Protein Derivative (PPD) tuberculin solution to trigger a delayed hypersensitivity reaction among those previously infected
• IGRA (IFN-y RELEASE ASSAY)


  Modern alternative for TST
• Xpert MTB/RIF


  Rapid test that detects M. tuberculosis and Rifampin-resistance
• DSSM (Direct Sputum Smear Microscopy)


  • Serves as one of the bases for categorizing TB cases
  • Used to monitor progress of patients with TB while they are on anti-TB treatment
  • Used to confirm cure at the end of treatment
• Sputum collection for diagnosis

  1. 3 sputa within 2 days

  2. 1st day: 1st specimen - any time of the day

  3. 2nd day: 2nd specimen - early morning (about 2 hrs after waking up), 3rd specimen - about 3-4 hrs after the 2nd specimen
• AFB smear reporting using Fuchsin stain


  • 0 in 300 fields - No AFB seen
  • 1-2 per 300 fields - Indeterminate (request new specimen for repeat testing)
  • 1-9 per 100 fields - 1+
  • 1-9 per 10 fields - 2+
  • 1-9 per 1 field - 3+
  • More than 9 per 1 field - 4+
• 2 out of three specimens should be positive to consider a patient positive for AFB
• Principle of acid-fast stain

  Acid-fast organisms are very hard to stain, but once stained, they are hard to decolorize. They appear RED in a blue background.
• Smear preparation and scanning


  Examined carefully by scanning at least 300 oil immersion fields (equivalent to 3 full horizontal sweeps of a 2cm long,1 cm wide smear)
• Acid-fastness


  Due to the presence of Mycolic Acid or Hydroxymethoxy acid or B-hydroxy butyric acid in the cell wall which is a long chain fatty acids with 50 – 90 carbon atoms
• Steam


  Used to get the carbol fuchsin primary dye to go into the cell wall. Once in, it will not come out
• Acid-alcohol decolorizer


  Will take the primary dye out of the non-acid fast cell walls since the primary dye does not bind strongly to the cell wall
• Acid-fast bacteria


  • Gram-positive, but in addition to peptidoglycan, the outer membrane or envelope of the acid-fast cell wall of contains large amounts of glycolipids, especially mycolic acids that make up approximately 60% of the acid fast cell wall in the genus Mycobacterium
• Layers of the acid-fast cell wall

  • Layer 1: Layer of peptidoglycan
  • Layer 2: Layer of arabinogalactan (a long carbohydrate composed of the sugars arabinose and galactose)
  • Layer 3: Outer membrane containing mycolic acids
  • Layer 4: Outer surface is studded with surface proteins that differ with the strain and species of the bacterium
• Other microorganisms that appear partially acid fast

  • Nocardia
  • Rhodococcus
  • Legionella micdadei
  • cysts of Cryptosporidium and Isospora
• AFB Staining Techniques


  • Fluorochrome
  • Ziehl-Neelsen
  • Kinyoun
• Other Methods of Acid Fast Staining

  • Cooper's Modification
  • Gabbett Modification
  • Muller-Chermock Carbol fuchsin Tergitol Cold Stain
  • Pappenheim's Method
  • Baumgarten's Method

  
• Modified Ziehl Neelsen stain

  Improved the detection of organisms in CSF and of intracellular organisms in WBC
• Fluorochrome stains (Truant Fluorochrome Stain)


  1. Fast

  2. LPO is used in the examination, slides can be used for routine acid fast staining

  3. More sensitive than the conventional carbolfuchsin stains because the fluorescent bacilli stand out brightly against the background
• Fluorochrome stain components


  • A - Auramine orange
  • A - Acid alcohol
  • P - Permanganate or Acridine orange
• Fluorochrome stain result


  • Primary stain will FLUORESCE when bacteria are properly excited rather than being red
  • Tissue debris and other background material is washed free of fluorescing dye with acid alcohol
  • Residual fluorescence of background debris may be minimized by a counterstain potassium permanganate or may be changed to a contrasting background by using acridine orange
• Fluorochrome stain methods


  • Auramine O Fluorochrome Staining
  • Richards-Miller Fluorescence Microscopy
  • Silver-Sonnenworth-Alex Method
• Ziehl-Neelsen Staining


  Mycobacterium spp. appear red or have a red-blue beaded appearance, whereas nonmycobacteria appear blue

  
• Ziehl-Neelsen Staining Procedure


  1. Sputum smear

  2. Heat fixing

  3. Carbol fuchsin

  4. Steam

  5. Acid alcohol

  6. Methylene blue
• Steaming


  Most crucial step in AFB staining

  
• Kinyoun acid-fast stain


  • Similar to Ziehl-Neelsen staining but no heat is used
  • Typical AFB appear as purple to red, slightly curved, short or long rods (2 to 8 μm); they also may be beaded or banded
  • Some nontuberculous species, such as MAC, appear pleomorphic and usually coccoid