Modified Thayer Martin Agar- Composition, Principle, Preparation, Results, Uses

Chocolate agar is a type of enriched growth medium that is used for the isolation of pathogenic bacteria, especially those that are fastidious and require special nutrients or factors to grow. Chocolate agar is a variant of blood agar, which contains red blood cells that have been lysed by heating to 80°C. The lysis of red blood cells releases hemoglobin, hemin (X factor), nicotinamide adenine dinucleotide (NAD or V factor), and other nutrients that are essential for the growth of bacteria such as Haemophilus influenzae and Neisseria meningitidis.

Modified Thayer Martin Agar is a selective medium that contains the following ingredients:

• GC Agar Base: This is the basic medium that provides nitrogenous nutrients in the form of casein and meat peptones, sodium chloride for osmotic balance, dipotassium phosphate and monopotassium phosphate for pH buffering, and corn starch for neutralizing toxic fatty acids that may be present in the agar. It also contains dextrose as a source of energy for the growth of gonococci.
• Hemoglobin Solution: This is added to the GC agar base to provide X factor (hemin), which is required by pathogenic Neisseria for their growth. Hemoglobin is derived from bovine blood and is sterilized by autoclaving.
• IsoVitaleX Enrichment: This is a defined supplement that provides V factor (nicotinamide adenine dinucleotide, NAD), vitamins, amino acids, coenzymes, dextrose, ferric ion, and other factors that improve the growth of pathogenic Neisseria. It is filter sterilized and added to the medium after cooling.
• VCNT Antibiotic Solution: This is a mixture of four antimicrobial agents that inhibit the growth of most other bacteria and fungi that may contaminate the specimens. The antibiotics are vancomycin, colistin, nystatin, and trimethoprim lactate (VCNT). Vancomycin inhibits gram-positive bacteria, colistin inhibits gram-negative bacteria including commensal Neisseria species, nystatin inhibits Candida albicans, and trimethoprim lactate inhibits Proteus species. The antibiotic solution is filter sterilized and added to the medium after cooling.

Modified Thayer Martin Agar is prepared by adding selective agents and supplements to a GC agar base. The preparation involves the following steps:

1. Add components of GC medium base into distilled water and bring volume to 730.0ml. Mix well thoroughly. Gently heat until boiling. Autoclave at 121°C for 15 minutes. Cool to 45-50°C.
2. Add hemoglobin to distilled water and bring volume to 250.0ml. Mix thoroughly. Autoclave at 121°C for 15 minutes. Cool to 45-50°C.
3. Add components of IsoVitaleX enrichment to distilled water and bring volume to 10.0ml. Mix thoroughly. Filter sterilize.
4. Add components of VCNT antibiotic solution to distilled water and bring volume to 10.0ml. Mix thoroughly. Filter sterilize.
5. To 730.0ml of cooled, sterile GC agar base, aseptically add 250.0ml of sterile hemoglobin solution, 10.0ml of IsoVitaleX enrichment, and 10.0ml of VCNT antibiotic solution. Mix thoroughly and pour into sterile Petri dishes or distribute into sterile tubes.

Modified Thayer Martin Agar is a selective medium that allows the growth of pathogenic Neisseria species while inhibiting most of the normal flora of bacteria and fungi that may be present in the specimens. The medium is based on Chocolate Agar, which is a GC Agar base enriched with bovine hemoglobin and IsovitaleX supplement. The selective property of Modified Thayer Martin Agar is achieved by adding four antibiotics to the medium: vancomycin, colistin, nystatin, and trimethoprim lactate. By using Modified Thayer Martin Agar, pathogenic Neisseria species can be isolated from specimens containing mixed flora of bacteria and fungi.

The colonies of pathogenic Neisseria species can be observed on Modified Thayer Martin Agar after 24-48 hours of incubation at 35-37°C in a CO2-enriched atmosphere. The typical colonial morphology on this medium is as follows:

• Neisseria gonorrhoeae: Small, grayish-white to colorless, mucoid with a smooth consistency and defined margins, and are typically 0.5 – 1.0 mm in diameter.
• Neisseria meningitidis: Medium to large, blue-gray, mucoid.

Other bacteria and fungi that may be present in the specimen are inhibited by the selective agents in the medium. However, some non-pathogenic Neisseria species may grow on Modified Thayer Martin Agar with reduced colony size and pigmentation. Therefore, additional biochemical and serological tests are recommended for the final confirmation of the identity of the isolates.

Modified Thayer Martin Agar is used for the isolation and cultivation of pathogenic Neisseria species, especially Neisseria gonorrhoeae and Neisseria meningitidis. These bacteria are the causative agents of gonorrhea and meningitis, respectively, and can cause serious infections in humans. Modified Thayer Martin Agar is useful for the detection of these pathogens from clinical specimens that contain mixed flora of bacteria and fungi. It can also be used for the subculture of pathogenic Neisseria from non-selective media.

• Modified Thayer Martin Agar may inhibit some strains of pathogenic Neisseria. Therefore, additional biochemical and serological tests are recommended for the final confirmation of the isolates.
• Culture for pathogenic Neisseria should be incubated in 3-7% CO2. Higher concentrations of CO2 may be inhibitory to some strains.
• Selective media for pathogenic Neisseria may suppress other pathogenic bacteria, such as Haemophilus, that may be present in the specimens.
• While "saprophytic" Neisseria are generally suppressed by selective media, the occasional recovery of N. lactamica on Modified Thayer Martin Agar has been reported.
• Some strains of Capnocytophaga species may grow on these selective media when inoculated with oropharyngeal specimens.

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