Middlebrook 7H9 Broth: Introduction, Composition, Principle, Procedure, Result-Interpretation, Uses, and Keynotes

Introduction

Middlebrook 7H9 broth is a liquid growth medium first described in 1958 by Middlebrook and Cohn for the specialized cultivation of Mycobacterium species. It is widely used in clinical and research settings for rapid growth, biochemical testing, and preparing inocula for drug susceptibility testing (DST).

This broth was developed to overcome the limitations of solid egg-based media (like Lowenstein-Jensen), offering significantly faster growth (4–14 days) and better suitability for automated systems.

Composition (Classical Formula per Liter)

The medium is composed of a basal salt solution and essential organic growth factors.

Ingredient	Amount	Function
Ammonium Sulfate	0.5 g	Nitrogen source
Disodium Phosphate	2.5 g	Buffering agent
Monopotassium Phosphate	Carbon source/wetting agent	Buffering agent
Sodium Citrate	0.1 g	Holds cations in solution
Magnesium Sulfate	0.05 g	Source of trace ions
L-Glutamic Acid	0.5 g	Nitrogen and carbon source
Ferric Ammonium Citrate	0.04 g	Iron source for growth
Biotin & Pyridoxine	0.5 mg / 1 mg	Essential vitamins/growth factors
Glycerol or Tween 80	2.0 ml / 0.5 g	Carbon source / wetting agent
ADC Enrichment	100 ml	Albumin, Dextrose, and Catalase

Principle

The medium relies on a precise balance of inorganic salts and organic supplements.

• Enrichment: The Middlebrook ADC Enrichment provides Dextrose for energy, Albumin to bind and neutralize toxic fatty acids, and Catalase to eliminate toxic peroxides.
• Wetting Agents: Tween 80 (Polysorbate 80) is often added to reduce surface tension, promoting homogenous growth and preventing the natural clumping of mycobacteria.

Procedure

1. Preparation: Suspend the base powder in water with glycerol or Tween 80, autoclave at 121°C for 10 minutes, cool to 45°C, and then aseptically add the ADC enrichment.
2. Inoculation: Inoculate the medium with a processed clinical specimen (e.g., decontaminated sputum) or a pure culture.
3. Incubation: Incubate at 35–37°C in an atmosphere of 5–10% CO2 for up to 8 weeks.

Result Interpretation

• Positive: Visible turbidity, granular sediment, or surface pellicle indicates growth. In automated MGIT systems, growth is detected via fluorescence caused by oxygen depletion.
• Negative: No turbidity after 8 weeks of incubation.
• Identification: All growth must be confirmed as M. tuberculosis complex or Nontuberculous Mycobacteria (NTM) using methods like Acid-Fast staining or nucleic acid probes.

Uses

• Primary Isolation: Cultivating mycobacteria from clinical specimens.
• Subculturing: Maintenance of laboratory stock strains.
• DST: Preparation of standardized inocula for drug susceptibility testing.
• Biochemical Tests: Used for assays like the tellurite reduction test.

Keynotes

• M. bovis Warning: This species is inhibited by glycerol; use Tween 80 instead for its cultivation.
• Safety: Work with virulent strains like M. tuberculosis requires Biosafety Level 3 (BSL-3) containment.
• Light Sensitivity: The prepared medium should be protected from direct light to prevent deterioration.

Further Readings

1. https://www.sciencedirect.com/topics/immunology-and-microbiology/middlebrook-7h9-broth
2. https://www.liofilchem.net/login/pd/ts/24436_TS.pdf
3. https://documents.thermofisher.com/TFS-Assets/LSG/manuals/IFU61344.pdf
4. https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU454012.pdf
5. https://hardydiagnostics.com/media/assets/product/documents/Middlebrook7H9Broth.pdf
6. https://cdn.media.interlabdist.com.br/uploads/2021/01/Middlebrook-7H9-Broth.pdf
7. https://en.wikipedia.org/wiki/Middlebrook_7H9_Broth
8. https://ntep.in/node/3988/CP-preparation-selective-middlebrook-7h9-liquid-medium
9. https://www.bd.com/en-us/products-and-solutions/products/product-families/bd-bactec-mgit-mycobacterial-growth-indicator-tubes
10. https://www.sigmaaldrich.com/US/en/product/sial/m0178
11. https://www.sigmaaldrich.com/MX/en/product/sial/m0178?srsltid=AfmBOootsA2qUytrg8uPb7ro3ezut2_mAxOXgu5j96UKa9LgV3WawccY
12. https://documents.thermofisher.com/TFS-Assets%2FMBD%2FInstructions%2FIFU9556.pdf