Thayer and Martin developed an improved version of Chocolate Agar by incorporating the antimicrobics vancomycin, colistin, and nystatin.
These additions were designed to minimize the overgrowth of contaminants, suppress the growth of saprophytic Neisseria species, and promote the growth of pathogenic Neisseria.
Later, Martin and Lester further enhanced the formulation by introducing the antibiotic trimethoprim, which increased the medium’s selectivity and proved useful in suppressing Proteus species.
Trimethoprim lactate specifically prevents the swarming of Proteus.
The modified medium, known as Modified Thayer Martin, serves as a selective and enriched culture medium.
It is specifically used for the isolation and cultivation of Neisseria species from mixed microbial populations.
The formulation effectively suppresses the growth of most other gram-negative diplococci, gram-negative bacilli, gram-positive organisms, and yeast.
Composition of Modified Thayer Martin Agar
Casein peptone – 7.5 g
Meat peptone – 7.5 g
Corn starch – 1.0 g
Dipotassium phosphate – 4.0 g
Monopotassium phosphate – 1.0 g
Sodium chloride – 5.0 g
Agar – 12.0 g
Hemoglobin solution – 10.0 g
IsovitaleX enrichment – 10.0 ml
Vancomycin – 300 μg
Colistin – 750 μg
Nystatin – 12,50 U
Trimethoprim – 5.0 mg
Demineralised water – 1000.0 ml
Final pH = 7.2 ± 0.2 at 25°C
IsovitaleX enrichment
Dextrose – 100.0 g
Vitamin B12 – 0.01 g
L-Glutamine – 10.0 g
Thiamine HCl – 0.003 g
L-Cystine – 1.1 g
Adenine – 1.0 g
p-Aminobenzoic acid – 13.0 g
Cocarboxylase – 0.1 g
Cystine HCl – 25.9 g
Guanine HCl – 0.03 g
NAD – 0.25 g
Ferric nitrate – 0.02 g
Demineralized water – 1000.0 ml
Preparation of Modified Thayer Martin Agar
Preparation of GC Agar base
Dissolve the components of the GC medium base in distilled water and adjust the final volume to 730.0 ml. Mix thoroughly, then heat gently until boiling. Sterilize by autoclaving at 121°C for 15 minutes. Allow the medium to cool to 45–50°C.
Preparation of Hemoglobin solution
Dissolve hemoglobin in distilled water and adjust the final volume to 250.0 ml. Mix well, sterilize by autoclaving at 121°C for 15 minutes, and cool to 45–50°C.
Preparation of IsoVitaleX enrichment (10 ml)
Dissolve all components in distilled water and adjust the volume to 10.0 ml. Mix thoroughly and sterilize by filtration.
Preparation of VCNT antibiotic solution (10 ml)
Dissolve the components in distilled water and make the final volume up to 10.0 ml. Mix well and sterilize by filtration.
Final preparation of the medium
To 730.0 ml of sterile, cooled GC agar base, aseptically add 250.0 ml of sterile hemoglobin solution, 10.0 ml of IsoVitaleX enrichment, and 10.0 ml of VCNT antibiotic solution. Mix thoroughly and dispense into sterile Petri plates or sterile tubes.
Principle of Modified Thayer Martin Agar
Modified Thayer Martin Agar is formulated on the basis of Chocolate II Agar, which includes an improved GC Agar base, bovine hemoglobin, and enrichment.
The GC II base supplies nitrogenous nutrients through casein and meat peptones, while phosphate buffer helps to maintain a stable pH.
Corn starch is included to neutralize toxic fatty acids that may be present in the agar.
Dextrose is incorporated to promote the growth of gonococci.
Hemoglobin serves as a source of the X factor (hemin).
IsovitaleX enrichment acts as a defined supplement providing the V factor (nicotinamide adenine dinucleotide, NAD), along with vitamins, amino acids, coenzymes, dextrose, ferric ion, and other growth-enhancing elements necessary for pathogenic Neisseria.
The selective nature of the medium is due to antimicrobial agents—vancomycin, colistin, and nystatin (collectively referred to as the V-C-N inhibitor)—which function to suppress the growth of normal flora.
Vancomycin is mainly effective against gram-positive bacteria.
Colistin inhibits gram-negative bacteria, including Pseudomonas species.
The presence of nystatin has been shown to inhibit Candida albicans.
Trimethoprim lactate is incorporated to prevent the swarming of Proteus species.
Result Interpretation on Modified Thayer Martin Agar
Neisseria gonorrhoeae colonies appear small, grayish-white to colorless, mucoid, with smooth consistency and clearly defined margins, typically measuring 0.5–1.0 mm in diameter.
Neisseria meningitidis colonies are medium to large in size, blue-gray in appearance, and mucoid in texture.
Uses of Modified Thayer Martin Agar
It is employed for the isolation of pathogenic Neisseria from specimens containing mixed bacterial and fungal flora.
It is specifically used for the isolation and cultivation of Neisseria species.
Limitations of Modified Thayer Martin Agar
Additional biochemical and serological tests are necessary for the final confirmation of results.
Cultures of pathogenic Neisseria should be incubated in an atmosphere containing 3–7% CO₂, as higher concentrations may inhibit the growth of certain strains.
The selective medium designed for pathogenic Neisseria may also suppress the growth of other pathogenic bacteria, such as Haemophilus.
Reports exist of N. gonorrhoeae strains being inhibited by components of the V-C-N inhibitor and by trimethoprim lactate.
Although selective media generally suppress “saprophytic” Neisseria, occasional recovery of N. lactamica on Thayer-Martin Selective Agar has been documented.
Certain strains of Capnocytophaga species may grow on these selective media, particularly when inoculated with oropharyngeal specimens.
References
Thayer, J.D., Frank, P.F., & Martin, J.E. Jr. (1965). Thayer-Martin selective medium for cultivating Neisseria meningitidis from the nasopharynx. American Journal of Public Health and the Nation’s Health, 55(6), 923–927.
Atlas, R.M., & Snyder, J.W. (2014). Handbook of Media for Clinical and Public Health Microbiology. CRC Press, Taylor & Francis Group, LLC, pp. 419–420.
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