THAYER MARTIN AGAR: FOUNDATION, PREPARATION AND USES - BIOLOGY - 2024

The agar Thayer Martin it is a highly nutritious and solid selective medium for the isolation of Neisseria meningitidis and Neisseria gonorrhoeae; both known as pathogenic or clinically important Neisserias.

One of the most important special characteristics of Thayer Martin Agar is its high content of nutritional supplements. This property is essential, because Neisserias are very demanding microorganisms from the nutritional point of view and therefore do not grow in common media.

Thayer Martin / Agar Thayer Martin business medium. Source Image 1: Taken by the author MSc. Marielsa Gil, image 2. Pixinio.com bacteriacolonized, modified, Thayer, Martin, agar.

On the other hand, since these microorganisms are normally found in non-sterile areas, the addition of inhibitors is necessary to prevent the growth of the accompanying flora, without affecting the development of the Neisseria genus.

This agar is composed of GC base agar, hemoglobin, additional nutritional supplements and a complex of inhibitory substances (antibiotics and antifungals). The commercial houses sell each of the supplements separately.

The sample to be sown in this medium will depend on the microorganism that is being sought. For Neisseria gonorrhoeae the ideal specimens are vaginal and urethral secretions. While for Neisseria meningitidis the most used samples are CSF, pharyngeal and nasopharyngeal exudates.

Basis

Neisserias are microorganisms classified as annoying and therefore their isolation is difficult. Therefore, Thayer Martín is a complex medium and each of its components fulfills a function that is explained below:

GC base agar

GC agar contains proteose peptone, cornstarch, sodium chloride, dipotassium phosphate, monopotassium phosphate, and agar-agar. Its components provide the basic nutrients for microbial development, neutralize toxic fatty acids, help maintain osmotic balance, define the pH and provide a solid consistency to the medium.

Hemoglobin

Hemoglobin provides factors V and X (nicotinamide adenine dinucleotide NAD and hemin respectively). For this reason, Haemophilus species also grow in this medium. Hemoglobin can be obtained commercially in a dehydrated form or fresh defibrinated bovine blood can be added to the medium.

Enrichment supplement

On the other hand, an enrichment supplement must also be added to the Thayer-Martin medium, since the nutrients contained in the base agar are not sufficient for the requirements of the Neisseria genus.

The most commonly used enrichment supplement is called isovitalex. It contains glutamine, adenine, NAD, cocarboxylase, guanine, ferric nitrate, p-amino benzoic acid, vitamin B12, thiamine, and glucose. All these compounds are necessary for the proper development of pathogenic Neisserias.

Inhibitors

As it is a highly nutritious medium, inhibitors must be used that prevent the growth of microorganisms from the usual flora of the area and thus favor the isolation of the Neisseria genus.

The inhibitor complex is composed of vancomycin, colistin, and nystatin. Vancomycin inhibits the growth of Gram positive bacteria, colistin prevents the growth of Gram negative bacteria such as Pseudomonas and some saprophytic Neisserias, and nystatin acts on yeasts such as Candida albicans.

However, Thayer Martin Agar was later modified; the changes consisted of the addition of trimethoprim, the increase in the amount of agar and the addition of extra glucose. These changes significantly improved the recovery of Neisseria gonorrhoeae species.

It should be noted that trimethoprim inhibits the growth of the genus Proteus and its consequent swarming formation. In this sense, it is the antibiotics that provide the selective character to the Thayer Martin medium.

Preparation

Original Thayer Martin agar

-GC agar

Weigh out 8.2 g of dehydrated GC medium and suspend in 100 ml. Mix and boil for 1 minute with frequent stirring to dissolve completely. Sterilize the mixture in an autoclave at 121 ° C for 15 minutes.

- 2% hemoglobin

Suspend 2 g of dehydrated hemoglobin in 2 or 3 ml of hot distilled water until a uniform mixture is formed. Add little by little more water to complete a volume of 100 ml. The suspension must be homogeneous before sterilizing.

Sterilize in autoclave for 15 minutes.

-Enrichment supplement

Reconstitute the commercial vial with the diluent provided by the same commercial company. Mix well. The amount of diluent to use will be specified in the kit instructions.

-VCN inhibition supplement (vancomycin, colistin, nystatin)

Reconstitute the vial with the diluent provided by the commercial company. Mix well. The amount of diluent to use will be specified in the kit instructions.

-Prepared

For every 100 ml of GC agar proceed as follows:

When the GC agar leaves the autoclave, allow it to cool to a temperature of approximately 50 ° C and add 2 ml of the prepared hemoglobin solution, 2 ml of the enrichment supplement (isobitalex or britalex), and 2 ml of the inhibition supplement. Mix and serve in sterile Petri dishes.

Let solidify and store in the fridge until use.

The color of the prepared medium is cherry red. The final pH of the medium is 7.2 ± 0.2

Modified Thayer Martin agar

Weigh out 8.2 g of dehydrated GC medium and suspend in 100 ml. Add 1 g of agar-agar and add 0.3 g of glucose. Mix and boil for 1 minute with frequent stirring to dissolve completely. Sterilize the mixture in an autoclave at 121 ° C for 15 minutes.

Prepare the hemoglobin and enrichment supplement as previously described.

The suppression supplement used is VCNT (vancomycin, colistin, nystatin, trimethoprim).

-Prepared

Proceed as described for the original Thayer Martin agar.

Use

Thayer Martin Agar should be allowed to warm before specimens are streaked. Commonly used samples are pharyngeal swabs, nasal swabs, vaginal, urethral, ​​and / or rectal discharge, and CSF.

Use fresh samples and make strong inoculums on the agar. The samples are seeded directly by material discharge and then exhaustion streaked onto the surface.

The plates are incubated at 35-37 ° C for 24 to 48 hours in a microaerophilic jar (5% CO ₂). At the end of the incubation time, the plates are examined for small, gray and sometimes mucoid-looking colonies.

Perform Gram and confirmatory biochemical tests on suspicious colonies.

QA

The following microbial strains can be used for quality control of Thayer Martin Agar.

Neisseria gonorrhoeae ATCC 49226 and Neisseria meningitidis ATCC 13090; satisfactory development is expected in both strains.

The following strains should also be included: Staphylococcus epidermidis ATCC 14990, Escherichia coli ATCC 25922, Proteus mirabilis ATCC 43071 and Candida albicans ATCC 10231. In all of them, a total or partial inhibition is expected in this medium.

Limitations

-We must bear in mind that bacteria that are resistant to the inhibitors used can grow in the medium.

-There are strains of Neisseria gonorrhoeae that can be sensitive to the concentration of vancomycin used. For this reason, it is recommended to use chocolate agar supplemented with isovitalex, but without inhibitors.

-There are strains of yeast resistant to nystatin that can grow in this medium and interfere with the isolation of pathogenic Neisserias, especially gonococci.

References

1. Valtek Diagnostic Laboratories. Thayer-Martin Agar. 2016. Available at: com
2. Britannia Laboratories. Thayer Martin Medium Modified. 2010.Available at: britanialab.com
3. Wikipedia contributors. Thayer-Martin agar. Wikipedia, The Free Encyclopedia. October 26, 2017, 16:33 UTC. Available at: wikipedia.org 4. Accessed February 28, 2019.
4. Britannia Laboratories. GC agar. 2010.Available at: britanialab.com.
5. Laboratories BBL ™ Medium Enrichment for Fastidious Microorganisms. 1999. Available in: bd.com
6. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.