Thayer and Martin have reported improvement of the Chocolate Agar formulation that contained antimicrobics vancomycin and colistin, Nystatin. These ingredients were added to limit the growth of contaminants, inhibit the development of the saprophytic Neisseria sp. and to enhance the development of the pathogenic Neisseria. Martin and Lester have added an antibiotic, trimethoprim, which made them more selective. They also demonstrated to be effective in the fight against Proteus spp. Trimethoprim lactate refrains Proteus swarming. The resultant medium is known as Modified Thayer Martin. It’s a suitable and rich medium to isolate as well as cultivation of the Neisseria species. from mixed flora and suppression of other Gram-negative diplococci and gram-negative bacilli yeast and other gram-positive organisms.
Composition of Modified Thayer Martin Agar
| Ingredients | Amount |
| Casein peptone | 7.5g |
| Meat peptone | 7.5g |
| Corn Starch | 1.0g |
| Dipotassium Phosphate | 4.0g |
| Monopotassium Phosphate | 1.0g |
| Sodium Chloride | 5.0g |
| Agar | 12.0g |
| Hemoglobin solution | 10.0g |
| IsovitaleX enrichment | 10.0ml |
| Vancomycin | 300μg |
| Colistin | 750μg |
| Nystatin | 12,50 U |
| Trimethoprim | 5.0mg |
| Demineralised water | 1000.0ml |
Final pH=7.2 ± 0.2 at 25°C
IsovitaleX enrichment
| Dextrose | 100.0g |
| Vitamin B12 | 0.01g |
| L-Glutamine | 10.0g |
| Thiamine HCl | 0.003g |
| L-Cystine | 1.1g |
| Adenine | 1.0g |
| p-aminobenzoic acid | 13.0g |
| Cocarboxylase | 0.1g |
| Cystine HCL | 25.9g |
| Guanine HCL | 0.03g |
| NAD | 0.25g |
| Ferric nitrate | 0.02g |
| Demineralized water | 1000.0ml |
Purpose
Modified Thayer Martin Medium Base utilized for the selective isolation and the enumeration of Neisseria species, particularly Neisseria gonorrhoeae
Principle of Modified Thayer Martin Agar
Modified Thayer Martin Agar is made from Chocolate II Agar and has a more refined GC Agar base bovine hemoglobin, and enrichment. This GC II base contains nitrogenous nutrients in the form of casein, meat peptones and phosphate buffers to keep the pH, and corn starch that neutralizes harmful acidic fatty acids present in the Agar. Dextrose is used to boost the growth of gonococci. Hemoglobin is a an X factors (hemin). IsovitaleX enhancer is supplement that contains V factor (nicotinamide Adenine Dinucleotide, NAD) as well as vitamins amino acids, coenzymes ferric ion, dextrose and other components that help to improve the growth rate of pathogenic Neisseria. The media that are targeted contain antimicrobial agents vancomycin colistin, and Nystatin (V-C-N inhibitor) to inhibit normal bacteria. Vancomycin is effective primarily against Gram-positive bacteria. Colistin blocks gram-negative bacteria which includes Pseudomonas species. Modified Thayer Martin Agar has this ingredient that has been shown to work in reduction of Candida albicans. Trimethoprim lactate stops the swarming of Proteus species.
Thayer-Martin’s medium is rich in antimicrobials which stop the growth of bacteria that are not N. Gonorrhoeae.
- vancomycin blocks gram-positive bacteria
- colistin blocks gram-negative bacteria such as those that cause commensal Neisseria species.,
- trimethoprim prevents swarming of Proteus Spp.,
- Nystatin is an antifungal drug.
The majority of Neisseria strains have complicated needs for growth. Gonococci are unable to expand on blood agars that are common to.
Preparation of Modified Thayer Martin Agar
- Preparation of the GC Agar base: Pour the components of GC medium base into the distilled water, and then increase the quantity to 730.0ml. Mix thoroughly. Warm gently until it is boiling. Autoclave at 121 degrees Celsius in 15 mins. Cool to 45-50degC.
- Preparation of Hemoglobin solution: Mix hemoglobin with the distilled water, and then increase the quantity to 250.0ml. Mix thoroughly. Autoclave at 121°C in 15 mins. Cool to 45-50degC.
- Preparation of IsoVitaleX enrichment (10ml): Add the components to water distilled and increase amount to 10.0ml. Mix thoroughly. Filter sterilize.
- Preparation of VCNT antibiotic solution (10ml): Add the components to distilled water and reduce the volume to 10.0ml. Mix thoroughly. Filter sterilize.
- Preparation of the medium: To 730.0ml of sterile, cooled GCC agar base, add 250.0ml of hemoglobin solution sterile. 10.0ml of IsoVitaleX enrichment , and 10.0ml of VCNT antibiotic solution. Mix thoroughly before pouring into sterilized Petri dishes or pour into tubes that are sterile.
Physical Properties of Modified Thayer Martin Agar
- Appearance: Cream to yellow homogeneous free flowing powder
- Gelling: Firm, comparable with 1.3% Agar gel.
- Colour and Clarity of prepared medium: The Basal Medium is a yellow transparent to slightly opalescent gel. Following the addition of haemoglobin or the sterile lysed blood, and any other ingredients chocolate colored opaque gel forms inside Petri plates.
- Reaction: Reaction of 4.2% w/v aqueous solution at 25°C. pH : 7.0±0.2
- pH: 6.80-7.20
Cultural Response
M413: Cultural traits discovered with added sterilelysed blood/Haemoglobin solutions (FD022), Vitamino Growth Supplement (FD025) and V.C.N. Supplement (FD023)/V.C.N.T. Supplement (FD024) after an incubation of 35-37°C for 18 to 48 hours
Result Interpretation of Modified Thayer Martin Agar
The typical colonial morphology of the media are as the following:
- Neisseria gonorrhoeae: Small, mucoid that ranges from grayish white to colorless with smooth consistency and distinct margins. They typically measure 0.5 to 1.0 millimeters in size.
- Neisseria meningitidis: Medium-sized to huge, blue-gray mucoid.
Culture and Isolation
- The specimen (urethral or endocervical) is directly injected into the plates used for culture (swabs made with wire or plastic shafts, and rayon Dacron and calcium alginate tips ) is used to make the specimen available for cultivation of gonococci).
- The inoculated plate must be placed immediately in an CO2-enriched (3 percentto 10 percent) environment and incubated for a period of 35o to 37oC.
- Small, transparent, grayish-white, glowing, colorless and smooth colonies can be seen.
Quality control
- Growing N. meningitidis strain for between 18 and 24 hours in MTM at 35-37degC , with 5 percent CO2 (or in the candle-jar).
- Check your MTM to determine specific colony shape.
- For sterility testing, place an uninoculated dish incubate for at least 48 hours at 35 to 37 degrees Celsius with a CO2 level of 5% (or in an jar that is a candle).
Additional Quality Control
- Examine the plates in accordance with “Product Deterioration.”
- Take a look at the plates in a visual way to make sure that any physical imperfections will not hinder the use.
- Calculate the pH potentiometrically the room temperature to ensure that it is in line with the standard of 7.2 + 0.2.
- Take note of the firmness of the plates during the process of inoculation.
- Incubate representative plates that have not been inoculated at 35 + 2°C for 72 hours and check for contamination by microbial species.
Application of Modified Thayer Martin Agar
- Thayer Martin Agar is used to aid in the identification from pathogenic Neisseria from samples containing mixed flora of both bacteria and fungi.
- It is utilized in the cultivation and isolation of the Neisseria species.
Limitations of Modified Thayer Martin Agar
- Further tests for serological and biochemical testing are suggested for final confirmation.
- Cultures of pathogenic Neisseria should be grown within CO2 containing 37. Higher levels of CO2 could hinder some strains.
- The selective media used for pathogenic Neisseria could also hinder other pathogenic bacteria e.g., Haemophilus.
- There are strains that have N. gonorrhoeae that are inhibited by the components of V-C.N. Inhibitor as well as trimethoprim lactate have been observed.
- Although “saprophytic” Neisseria are generally controlled by selective media, occasionally, the recuperation from N. lactamica on Thayer Martin Selected Agar is documented.
- Certain species of Capnocytophaga species can thrive on these media with a selective nature when they are infected using oropharyngeal crystals.
