Comparative Morphology of Acid-Fast Bacilli

¹ Department of Infectious Diseases, Medical School, University of California at Los Angeles., Western Trudeau Streptomycin Laboratory, Olive View, California.
² Department of Infectious Diseases, Medical School, University of California at Los Angeles.

A morphological study, both cultural and microscopic, was conducted, using ten strains of virulent and avirulent tubercle bacilli and two acid-fast saprophytic organisms isolated from sputum. The organisms were studied on five different media and preparations were made for microscopic examination using the following techniques: wet preparations with the ordinary light microscope, darkfield microscope and phase microscope; unstained dried films with ordinary light microscope and electron microscope; and fixed dried films stained with methylene blue, Gram stain and ZiehlNeelsen stain.

At least half a dozen different forms of acid-fast bacilli were clearly distinguished by colonial morphological characteristics. Virulent human types were distinguished from all other organisms by their dry, crumbling, spreading colonies on egg media and Bordet's agar, their characteristic large serpentine colonies on sodium oleate agar, and thin corded pellicles which grew up the walls of the tube in the serum-synthetic medium. The avirulent human type produced granular colonies with no crumbling on egg media and Bordet's agar and a small non-serpentine colony on sodium oleate agar. Thick folded pellicles in serum-synthetic media characterized this organism. The virulent bovine type also produced serpentine growth on sodium oleate agar and spreading crumbling colonies on egg media and Bordet's medium. BCG always produced flaky growth on egg media and characteristic umbilicated colonies on Bordet's medium. Avian organisms showed smaller colonies with little spreading and usually, a smooth surface. The saprophytes always produced smooth shiny colonies on all solid media and heavy smooth pellicles on serum-synthetic medium. Only virulent organisms produced spreading on egg media and Bordet's agar and serpentine flat spreading colonies on sodium oleate agar. On the latter medium, only virulent organisms produced satisfactory growth, the avirulent types growing very poorly.

Microscopically, the serpentine or corded arrangement of virulent tubercle bacilli contrasted sharply with the diffuse granular appearance of avirulent strains. Dense intracellular bodies, often bipolar, seen in all strains of acid-fast bacilli corresponded in form and position to similar structures in electron microscope films and in methylene blue stained smears. These nucleoids appeared to be quite different from the beading or granules often seen in acid-fast stained smears. Large fusiform swellings were seen only in saprophytic strains though small bulges occasionally appeared in other acid-fast bacilli. Saprophytes and avian tubercle bacilli also occasionally showed long filamentous rods and forms suggesting branching growth. Neither gross colonial nor microscopic morphological, nor tinctorial differences were noted between human type tubercle bacilli which were resistant to, or dependent on, streptomycin and their parent susceptible strains.