Culturing Mycobacterium tuberculosis Under Iron-Limited Conditions

Begin with an agar plate containing Mycobacterium tuberculosis colonies grown on a nutrient-enriched medium.

Inoculate a single colony into a nutrient-supplemented broth and incubate with agitation for uniform growth.

Allow the culture to reach the late logarithmic phase, where the cells are metabolically active.

Pipette a small volume onto an enriched agar plate, spread it evenly, and incubate until a continuous bacterial layer forms.

Moisten a sterile swab with a low-iron minimal medium (LIMM) and collect the bacteria.

Suspend them in a plastic container containing LIMM to avoid iron contamination.

Add a dispersing agent to prevent clumping and incubate without shaking.

Under iron-limiting conditions, bacteria activate stress responses and produce siderophores in the cytoplasm.

These are packaged into extracellular vesicles and released into the surroundings.

Siderophores capture iron and help bacteria survive.

This system highlights mycobacterial adaptation and survival under iron-limited conditions

Inoculate a single colony of MTB in two milliliters of mycobacterial broth medium supplemented with ADN enrichment. Incubate with agitation at 37 degrees Celsius. When there is visible growth,