Culturing Mycobacterium tuberculosis Under Iron-Limited Conditions

Abstract

Source: Gupta, S., et al. Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria. J. Vis. Exp. (2019)

This video demonstrates the procedure for culturing Mycobacterium tuberculosis under iron-limited conditions to induce iron stress and promote the secretion of siderophores that support bacterial survival.

Protocol

1. Growing Mycobacteria in Iron-limited Conditions

1. Thaw out 50 µL of a frozen 15% glycerol stock of Mtb (Mycobacterium tuberculosis) and streak an agar plate supplemented with 10% ADN (Albumin – Dextrose – NaCl) enrichment (5 g/L albumin, 2 g/L dextrose, and 0.85 g/L sodium chloride, 0.2% glycerol, and 0.05% Tween-80). Incubate the plate at 37 °C until colonies are visible.
2. Inoculate a single colony of Mtb in 2 mL of mycobacterial broth medium (Table of Materials) supplemented with ADN enrichment. Incubate with agitation at 37 °C.
3. Let the Mtb culture grow to the late logarithmic phase (OD₅₄₀ is ~0.8). To check this, measure the OD₅₄₀ using a spectrophotometer.
4. Spread 200 µL of the late logarithmic culture onto the mycobacterial agar plates (Table of Materials) supplemented with 0.2% glycerol, 0.05% Tween-80, and ADN. Inoculate at least 5 plates. Incubate the plates at 37 °C until bacterial growth is visible as a confluent layer. This takes ~1 week for Mtb.
5. Wet a sterile cotton swab in LIMM (low-iron minimal medium). Use this swab to collect bacteria from the agar plates and inoculate 100 mL of LIMM to prepare a concentrated bacterial suspension with an OD₅₄₀ of ~1.0.
6. Dilute this suspension 10 times to 1 L with LIMM and divide it into two 2 L sterile plastic bottles, each containing 500 mL of culture.
7. Take out 2 mL of culture and transfer it to a 5 mL culture tube. Add 10 µL of 10% vol/vol tyloxapol.
8. Incubate the cultures at 37 °C for 14 days.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Middlebrook 7H10 Agar	BD Difco	262710	Mycobacterial agar plates
Middlebrook 7H9 Broth	BD Difco	271310	Mycobacterial broth medium
Polypropylene thin walled centrifuge tube 13x15 mm	Beckman Coulter	344059	Polypropylene thin walled centrifuge tube 13x15 mm
Vacuum filtration flasks	CellPro	V50022	Filter Unit