This study investigates the survival mechanisms of Mycobacterium tuberculosis under iron-limited conditions. It highlights the role of siderophores in iron acquisition and bacterial adaptation.
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, extract two milliliters of the culture and measure the OD 540 on a spectrophotometer.
Stop the incubation when the OD 540 reaches 0.8 which indicates the late logarithmic phase. Spread 200 microliters of the late logarithmic culture onto the mycobacterial agar plates supplemented with 0.2 glycerol, 0.5% Tween 80, and ADN. Inoculate at least five plates.
Incubate the plates at 37 degrees Celsius. And after one week, the bacterial growth is visible as a confluent layer. Then wet a sterile cotton swab in a low iron minimal medium.
Use this swab to collect bacteria from the agar plates. Inoculate the bacteria in 100 milliliters of low iron minimal media in a tube. Collect sufficient bacteria to prepare a suspension with an OD 540 of one.
Dilute the suspension 10 times to one liter with low iron minimal medium and divide it into two sterile plastic bottles. Next, transfer two milliliters of the culture to a five milliliter culture tube. Add 10 microliters of 10% volume by volume tyloxapol.
Incubate the cultures at 37 degrees Celsius for 14 days.
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