Mycobacterium smegmatis is a bacterium frequently used in research laboratories. It belongs to the genus Mycobacterium, a group that includes human pathogens causing diseases such as tuberculosis and leprosy. Unlike its relatives, M. smegmatis is non-pathogenic. This fast-growing organism serves as a safe and efficient model for scientists to study the unique biology of the entire genus.
Morphology and Basic Structure
Under high magnification, M. smegmatis appears as a slender, rod-shaped cell (bacillus). Cells typically measure between 3.0 and 5.0 micrometers in length, placing them at the upper end of the size range for the genus Mycobacterium. These bacilli are generally observed singly or in small, disorganized clusters, sometimes exhibiting a slightly curved or bent appearance. They do not form the long, extensive chains seen in many other types of bacteria.
The complex cell wall structure of M. smegmatis provides resilience to the organism. This wall contains a high concentration of mycolic acids, which are long-chain fatty acids that create a thick, waxy, lipid-rich outer layer. This hydrophobic coat makes the cell wall highly impermeable, contributing to the bacterium’s resistance to many common disinfectants and conventional stains. This unique composition necessitates a specialized preparation technique before the organism can be clearly visualized under a microscope.
Preparing for Viewing: The Acid-Fast Stain
The waxy nature of the M. smegmatis cell wall prevents the uptake of standard aqueous stains. To overcome this barrier, a differential staining technique called the acid-fast stain, such as the Ziehl-Neelsen or Kinyoun method, is used. This method uses a primary stain, carbol fuchsin, which is a lipid-soluble phenolic dye that penetrates the mycolic acid layer. Penetration is often facilitated by heating the slide or by using higher concentrations of phenol in the cold Kinyoun method.
Once the primary dye has entered the cell, it is trapped within the cell wall’s dense lipid matrix. A subsequent step involves washing the slide with a strong acid-alcohol solution, which serves as a decolorizer. Non-acid-fast bacteria lose the primary stain immediately in the presence of the acid, but M. smegmatis cells retain the dye due to the mycolic acid barrier. This resistance to decolorization by acid is the origin of the term “acid-fast,” making the procedure a way to identify members of the genus Mycobacterium.
Visualizing Under Different Microscopes
When viewed through a bright-field light microscope after acid-fast staining, M. smegmatis presents a distinctive appearance. The cells that retained the carbol fuchsin stain appear as red or pink rods. These stained bacilli stand out sharply against the viewing field, which is counterstained blue with a dye like methylene blue. Because M. smegmatis often clumps together, the red rods may be seen in small clusters or tangled arrangements on the slide, resembling tiny, scattered matchsticks.
For a more detailed examination of the internal architecture, scientists use electron microscopy. Transmission Electron Microscopy (TEM) reveals the multi-layered structure of the cell wall that is responsible for its resistance. This technique can visualize the dense layer of mycolic acids, which appears as an electron-transparent zone just outside the cell membrane. TEM also allows for precise measurement of cellular components, showing that M. smegmatis has a larger cell volume and diameter compared to its pathogenic counterpart, M. tuberculosis.
Why Scientists Study M. smegmatis
M. smegmatis is an important “model organism” for research into mycobacterial disease. Its rapid growth rate, with colonies forming in just three days, contrasts sharply with the weeks or months required for pathogenic species. This allows for accelerated genetic and biochemical experiments that would be impractical with slow-growing bacteria.
Structurally and genetically, M. smegmatis shares thousands of homologous genes and the unique mycolic acid cell wall with pathogens. Researchers exploit this similarity to safely investigate processes, such as how the cell wall is synthesized and how genetic mutations affect its structure and function. Studying this microbe allows for the rapid screening of new drug compounds, like the tuberculosis drug Bedaquiline, which was initially identified through screens using M. smegmatis.