**Core Concept**
Mycobacterial species differentiation is crucial for clinical diagnosis and treatment planning. This involves identifying the specific genus and species of the pathogen, which can be done through various methods including morphological, biochemical, and molecular techniques.

**Why the Correct Answer is Right**
Mycobacterial species are differentiated based on their growth characteristics, such as colony morphology, temperature and pH requirements, and the presence of specific pigments. Additionally, biochemical tests like nitrate reduction and Tween 80 hydrolysis can help distinguish between different species. Molecular techniques like DNA sequencing and PCR can also be used to identify specific mycobacterial species.

**Why Each Wrong Option is Incorrect**
**Option A:** This option is incorrect because it does not accurately describe a method used for mycobacterial species differentiation. While some methods may involve multiple steps, the key point is that mycobacterial species are differentiated based on their unique characteristics.
**Option B:** This option is incorrect because it is a characteristic shared by many mycobacterial species, but it is not a specific method used for differentiation. Mycobacteria are indeed acid-fast, but this trait is not sufficient for differentiation.
**Option C:** This option is incorrect because it is a method used for bacterial identification in general, but it is not specific to mycobacterial species differentiation. While some mycobacteria may be identified through Gram staining, this is not a reliable method for differentiation.

**Clinical Pearl / High-Yield Fact**
Mycobacterial species can be differentiated based on their growth characteristics, biochemical reactions, and molecular features. It is essential to identify the specific species to choose the appropriate treatment regimen, as some mycobacteria may require specific antibiotics or treatment approaches.

**Correct Answer: D.**