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Microbiology

Major Pathogens

Identifying Mycobacteria and Nocardia involves a combination of techniques, starting with recognizing their unique characteristics. This section covers the key features used in initial identification of major pathogens like Mycobacterium tuberculosis, Mycobacterium avium intracellulare (MAC), Mycobacterium kansasii, Mycobacterium abscessus group, Mycobacterium marinum, and Nocardia species

General Principles

  • Acid-Fast Staining: This is the cornerstone of mycobacterial and Nocardia identification. Remember, the cell walls of these organisms contain mycolic acids, which make them resistant to decolorization with acid-alcohol after staining
  • Colony Morphology: The appearance of colonies on solid media (e.g., size, shape, color, texture) can provide clues to the identity of the organism
  • Growth Rate: Mycobacteria are generally slow-growing, but some species grow faster than others. Nocardia also grows slower than typical bacteria
  • Environmental Factors: Temperature, light, and the presence of specific nutrients can influence growth characteristics

Acid-Fast Reaction

  • Principle: The acid-fast stain uses a primary dye (carbolfuchsin), a decolorizer (acid-alcohol), and a counterstain (methylene blue or malachite green)
  • Procedure
    1. Smear Preparation: Prepare a thin smear of the specimen or culture on a glass slide
    2. Fixation: Heat-fix the smear to kill the bacteria and adhere them to the slide
    3. Staining: Flood the smear with carbolfuchsin and apply heat (Ziehl-Neelsen) or allow to stain for a longer period (Kinyoun)
    4. Decolorization: Decolorize with acid-alcohol until the smear is clear
    5. Counterstaining: Counterstain with methylene blue or malachite green
  • Interpretation
    • Acid-Fast Bacteria (AFB): Appear red or pink against a blue or green background
    • Non-Acid-Fast Bacteria: Appear blue or green
  • Reporting: Report the number of AFB observed per field (e.g., no AFB seen, 1-9 AFB/field, 1+, 2+, 3+)

Major Pathogens: Acid-Fast Reaction, Colony Morphology, and Growth Characteristics

Mycobacterium tuberculosis

  • Acid-Fast Reaction: Strongly acid-fast
  • Colony Morphology
    • Egg-based media (e.g., Löwenstein-Jensen): Rough, dry, wrinkled colonies with a buff or cream color (“cauliflower-like”)
    • Agar-based media (e.g., Middlebrook 7H10, 7H11): Flat, irregular colonies with a matte surface
  • Growth Characteristics
    • Slow-growing: Requires 2-6 weeks for visible colonies to appear
    • Optimal growth temperature: 35-37°C
    • Requires aerobic conditions
    • Niacin Production: Positive
    • Nitrate Reduction: Positive

Mycobacterium avium complex (MAC)

  • Acid-Fast Reaction: Acid-fast
  • Colony Morphology
    • Egg-based media: Smooth, transparent to opaque, dome-shaped colonies. Can be buff, cream, or even pinkish in color
    • Agar-based media: Similar to egg-based media, but may be more mucoid
  • Growth Characteristics
    • Slow-growing: Requires 7-21 days for visible colonies to appear
    • Optimal growth temperature: 35-37°C
    • Requires aerobic conditions
    • Niacin Production: Negative
    • Nitrate Reduction: Negative

Mycobacterium kansasii

  • Acid-Fast Reaction: Acid-fast
  • Colony Morphology
    • Egg-based media: Smooth or rough colonies with a distinct photochromogenic property (develops a yellow-orange pigment when exposed to light)
    • Agar-based media: Similar to egg-based media
  • Growth Characteristics
    • Relatively rapid-growing: Colonies may appear within 7-21 days
    • Optimal growth temperature: 35-37°C
    • Requires aerobic conditions
    • Niacin Production: Negative
    • Nitrate Reduction: Positive

Mycobacterium marinum

  • Acid-Fast Reaction: Acid-fast
  • Colony Morphology
    • Egg-based media: Smooth or rough colonies with a photochromogenic property (develops a yellow pigment when exposed to light)
    • Agar-based media: Similar to egg-based media
  • Growth Characteristics
    • Slow-growing: Colonies may appear within 7-21 days
    • Optimal growth temperature: 30-32°C (growth is inhibited at 37°C)
    • Requires aerobic conditions

Mycobacterium abscessus

  • Acid-Fast Reaction: Acid-fast
  • Colony Morphology
    • Agar-based media: Smooth or rough colonies, often with a matte surface. Can be buff or cream-colored
  • Growth Characteristics
    • Rapidly growing: Colonies appear within 3-7 days
    • Optimal growth temperature: 28-30°C
    • Grows on MacConkey agar without crystal violet
    • Tolerance to Salt: Can grow in up to 5% NaCl

Nocardia spp.

  • Acid-Fast Reaction: Weakly acid-fast (modified acid-fast stain may be required)
  • Colony Morphology
    • Agar-based media: Wrinkled, dry, chalky or velvety colonies. Can be white, cream, orange, or pink in color
  • Growth Characteristics
    • Relatively slow-growing: Colonies may appear within 3-7 days, but some species can take longer
    • Optimal growth temperature: 25-37°C
    • Requires aerobic conditions
    • Hydrolysis of Casein, Tyrosine and Xanthine: Positive

Key Terms

  • Acid-Fast Stain: A differential stain used to identify acid-fast bacteria, such as mycobacteria and some Nocardia species
  • Carbolfuchsin: A red dye used as the primary stain in the acid-fast staining procedure
  • Acid-Alcohol: A decolorizing agent used in the acid-fast staining procedure to remove the carbolfuchsin from non-acid-fast bacteria
  • Methylene Blue/Malachite Green: Counterstains used in the acid-fast staining procedure to stain non-acid-fast bacteria
  • Colony Morphology: The macroscopic appearance of bacterial colonies grown on solid media, including size, shape, color, texture, and edge characteristics
  • Growth Rate: The rate at which bacteria multiply and form visible colonies on solid media
  • Photochromogen: A microorganism that produces pigment when exposed to light
  • Löwenstein-Jensen (LJ) Medium: An egg-based medium commonly used for the cultivation of mycobacteria
  • Middlebrook 7H10/7H11 Agar: Agar-based media used for the cultivation of mycobacteria
  • MacConkey Agar: A selective and differential agar used to differentiate between Gram-negative bacteria based on their ability to ferment lactose
  • Aerobic: Requires oxygen for growth
  • Niacin Test: A biochemical test used to detect the production of niacin (nicotinic acid) by certain mycobacteria species
  • Nitrate Reduction Test: A biochemical test used to determine the ability of an organism to reduce nitrate to nitrite or other nitrogenous compounds
  • Modified Acid-Fast Stain: An acid-fast staining procedure that uses a weaker decolorizing agent to prevent over-decolorization of weakly acid-fast organisms, such as Nocardia species
  • Hydrolysis: The chemical breakdown of a compound due to reaction with water
  • Casein: A family of related phosphoproteins that are commonly found in mammalian milk, making up 80% of the proteins in cow’s milk and between 20% and 45% of the proteins in human milk
  • Tyrosine: Is one of the 20 standard amino acids that are used by cells to synthesize proteins
  • Xanthine: A purine base found in most human body tissues and fluids, as well as in other organisms