Middlebrook Agar- Composition, Principle, Preparation, Results, Uses


Middlebrook Agar is a type of culture medium that is specially designed for the isolation and cultivation of mycobacteria, which are a group of bacteria that cause diseases such as tuberculosis and leprosy. Mycobacteria are difficult to grow in conventional media because they have a complex cell wall that makes them resistant to many antibiotics and disinfectants. They also require specific nutrients and environmental conditions for optimal growth.

Middlebrook Agar was developed by Dubos and Middlebrook in the 1940s and 1950s, based on their earlier formulations that contained oleic acid and albumin as key ingredients. These components help to protect the mycobacteria from toxic agents and enhance their recovery on primary isolation. Later, Middlebrook and Cohn improved the formulation by adding various inorganic salts, glycerol, malachite green dye, and agar. The resulting medium, known as Middlebrook 7H10 Agar, was found to support faster and more luxuriant growth of mycobacteria than the egg-based media commonly used at that time.

Middlebrook Agar can be supplemented with different additives to enrich the medium or make it more selective for certain types of mycobacteria. For example, Middlebrook OADC Enrichment contains sodium chloride, dextrose, bovine albumin, catalase, and oleic acid, which provide essential electrolytes, carbon source, protection against oxidative stress, and fatty acids for mycobacterial metabolism. Middlebrook ADC Enrichment contains only albumin, dextrose, and catalase. Middlebrook PANTA Enrichment contains polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin, which inhibit the growth of most bacteria other than mycobacteria.

Middlebrook Agar is widely used for the isolation, cultivation, and sensitivity testing of Mycobacterium tuberculosis, the causative agent of tuberculosis. It can also be used for other Mycobacterium species, such as Mycobacterium leprae (leprosy), Mycobacterium avium complex (MAC), Mycobacterium kansasii (lung infections), and Mycobacterium marinum (fish tank granuloma). Middlebrook Agar allows for the observation of colony morphology, pigment production, and cord formation of mycobacteria, which are useful characteristics for identification.

Middlebrook Agar is one of the most important cultural media for mycobacteriology. It has several advantages over other media, such as:

  • It supports the growth of both slow- and fast-growing mycobacteria.
  • It reduces the contamination by other bacteria due to the presence of malachite green dye.
  • It allows for the detection of drug-resistant strains of mycobacteria by incorporating antibiotics into the medium.
  • It facilitates the differentiation of mycobacteria based on their phenotypic features.

However, Middlebrook Agar also has some limitations, such as:

  • It requires incubation in a 5-10% CO2 atmosphere for optimal growth of mycobacteria.
  • It may not recover all types of mycobacteria if the specimens are not properly decontaminated before inoculation.
  • It may not provide conclusive identification of mycobacteria without further biochemical, immunological, molecular, or mass spectrometry testing.

In this article, we will discuss the composition, principle, preparation, results, uses, and limitations of Middlebrook Agar in detail. We will also provide some examples of how Middlebrook Agar can be used in clinical microbiology laboratories.