Stain, Morph, Growth
This section discusses the analytic procedures related to acid-fast reaction, colony morphology, and growth characteristics for major pathogens, common contaminants, and less common Mycobacterium pathogens
The Big Picture: A Multi-Faceted Approach
Identifying Mycobacteria and Nocardia in the lab is rarely a “one-test-fits-all” situation. Instead, it’s a process of gathering clues from different sources and putting them together to arrive at an accurate identification. Three key areas provide these clues: the acid-fast reaction, colony morphology, and growth characteristics
Acid-Fast Reaction: The First Line of Defense
The acid-fast stain is a fundamental tool. Mycobacteria and Nocardia have waxy cell walls containing mycolic acids, which cause them to retain the primary dye (carbolfuchsin) even after treatment with acid-alcohol. This is what makes them “acid-fast.” While most members of these genera are acid-fast, the intensity can vary. For example, Nocardia may be weakly acid-fast, requiring a modified staining technique
Colony Morphology: Visual Inspection
Once a culture grows, observing the colonies provides further information. Key features include:
- Shape: Are the colonies round, irregular, or filamentous?
- Size: Are they pinpoint or large and spreading?
- Texture: Are they smooth, rough, wrinkled, or mucoid?
- Color: Are they buff, cream, yellow, orange, pink, or white? Some species are photochromogenic, meaning they produce pigment only when exposed to light, while others are scotochromogenic, producing pigment in the dark
Growth Characteristics: Understanding Growth Requirements
Mycobacteria are known for their slow growth, requiring specialized media and longer incubation times compared to typical bacteria. Important factors include:
- Growth Rate: How quickly do colonies appear? Rapid growers form colonies within a week, while slow growers may take several weeks
- Temperature: What is the optimal temperature for growth? Some species, like M. marinum, prefer lower temperatures
- Media Requirements: Do they require specific nutrients or supplements, such as iron for M. haemophilum?
- Atmosphere: Are they strict aerobes (requiring oxygen)?
Differentiating Key Players
- M. tuberculosis: Rough, dry, buff-colored colonies on egg-based media, slow growth, and positive niacin and nitrate reduction tests
- MAC (M. avium complex): Smooth, transparent to opaque colonies, slow growth
- M. kansasii: Photochromogenic colonies, relatively rapid growth, and positive nitrate reduction test
- M. marinum: Photochromogenic colonies, slow growth, and prefers lower temperatures
- M. abscessus: Rapidly growing, can grow on MacConkey agar, and often causes skin and soft tissue infections
- M. gordonae: A common contaminant, with deep yellow to orange scotochromogenic colonies
- Nocardia spp.: Weakly acid-fast, wrinkled, dry colonies, and relatively slow growth
- M. leprae: An obligate intracellular parasite that cannot be cultured on artificial media
- M. haemophilum: Requires iron supplementation for growth
- M. scrofulaceum: Causes scrofula, produces yellow to orange scotochromogenic colonies