What is Thayer Martin Medium?

• Thayer Martin Medium, also known as Thayer-Martin Agar, is a specialized culture medium developed for the isolation and identification of Neisseria gonorrhoeae and Neisseria meningitidis. It was first introduced by Carpenter and Morton as an improved medium to isolate Gonococci within 24 hours.
• The original GC (gonococcal) medium supplemented with haemoglobin and yeast concentrate was found to be effective in isolating gonococci. However, Thayer and Martin further developed the medium to enable the primary isolation of Neisseria gonorrhoeae and Neisseria meningitidis from specimens containing mixed microbial flora obtained from the throat, vagina, rectum, and urethra.
• Thayer and Martin incorporated Vancomycin, Colistin, and Nystatin into the medium. Vancomycin and colistin act as selective agents by inhibiting the growth of gram-positive and gram-negative bacteria, respectively. Nystatin is added to inhibit fungal growth. This medium may also inhibit Haemophilus species, while certain strains of Capnocytophaga species may still grow on it when inoculated with oropharyngeal specimens.
• The medium contains special peptone, which provides essential nutrients to the organisms. Starch is included to neutralize any toxic fatty acids that may be present in the agar. Hemoglobin serves as the X factor, and the V factor (N.A.D.) is provided by an added supplement. The supplement (FD025) not only supplies vitamins, amino acids, and coenzymes but also enhances the growth of pathogenic Neisseria species.
• Over time, the Thayer-Martin formulation underwent further modifications, leading to the development of Martin-Lewis Agar. Martin-Lewis Agar contains an increased level of vancomycin, making it even more effective in inhibiting gram-positive organisms. This modification has resulted in enhanced recovery of gonococci from clinical samples.
• Martin-Lewis Agar is a selective and enriched medium designed for the isolation and cultivation of Neisseria species from mixed flora. The incorporation of hemoglobin, Bio-X, and dextrose in the culture media provides an ample supply of nutrients, allowing for luxuriant growth of fastidious microorganisms.
• Overall, Thayer Martin Medium and its modified version, Martin-Lewis Agar, have played crucial roles in the isolation and identification of pathogenic Neisseria species, particularly Neisseria gonorrhoeae and Neisseria meningitidis, from clinical specimens containing complex microbial populations.

Composition of Martin Lewis Agar

Ingredients	Gms/L
Casein Peptone	7.5
Meat Peptone	7.5
Corn Starch	1.0
Potassium Phosphate, dibasic	4.0
Potassium Phosphate, monobasic	1.0
Sodium Chloride	5.0
Agar	10.0
Hemoglobin	10.0
Dextrose	1.5
Enrichment Bio-X	10 ml
Antibiotics V.C.A.T	10 ml

pH 7.2 +/- 0.2 at 25º C

Enrichment Bio-X

Purified Water Vitamin B12	0.01 g
Thiamine Pyrophosphate	0.1 g
L-Glutamine	10.0 g
Ferric Nitrate	0.02 g
Adenine	1.0 g
Thiamine Hydrochloride	0.003 g
Guanine Hydrochloride	0.03 g
L-Cysteine Hydrochloride	25.9 g
p-Aminobenzoic Acid	0.013 g
L Cystine	1.1 g
Nicotinamide Adenine Dinucleotide	0.25 g
Dextrose	100.0 g

Principle of Martin Lewis Agar

The principle of Martin-Lewis Agar lies in its composition and the use of specific ingredients to selectively support the growth of pathogenic Neisseria species while inhibiting the growth of unwanted microorganisms.

The agar base of Martin-Lewis Agar is derived from chocolate agar, which itself is an improved GC Agar base. This base provides essential nutrients in the form of casein and meat peptones, supplying nitrogenous compounds necessary for bacterial growth. It also contains a phosphate buffer to maintain the pH of the medium and cornstarch, which helps neutralize any toxic fatty acids that may be present in the agar.

Hemoglobin is added to the medium, serving as a source of the X factor, specifically hemin. The X factor is essential for the growth of certain fastidious microorganisms such as Neisseria species.

The chemically defined enrichments, specifically the Bio-X enrichment, play a crucial role in providing the V factor (nicotinamide adenine dinucleotide – NAD). The V factor is another essential growth factor required by pathogenic Neisseria species. Additionally, the Bio-X enrichment supplies vitamins, amino acids, coenzymes, dextrose, ferric ions, and other growth factors that further enhance the growth and optimal development of pathogenic Neisseria species.

Martin-Lewis Agar is also a selective medium due to the presence of specific antimicrobial agents. Vancomycin, primarily effective against gram-positive bacteria, acts as a selective agent by inhibiting the growth of these organisms. Colistin, on the other hand, targets gram-negative bacteria, including Pseudomonas species. Anisomycin is added to inhibit yeasts, and trimethoprim is included to suppress the growth of Proteus species.

By combining these components, Martin-Lewis Agar creates an environment that supports the growth of pathogenic Neisseria species while inhibiting the growth of unwanted microorganisms, including normal flora and certain gram-positive and gram-negative bacteria. This selective medium is valuable for the isolation and cultivation of pathogenic Neisseria species from mixed microbial populations, aiding in the accurate diagnosis and identification of these pathogens.

Intend to Use

Martin-Lewis agar is used to aid in the identification of pathogenic Neisseria strains from samples with a mix of flora from both fungi and bacteria.

Preparation of Martin Lewis Agar

The preparation of Martin-Lewis Agar involves several steps to ensure the proper incorporation of the ingredients and the sterilization of the medium. Here is a summary of the preparation process:

1. Begin by suspending 36 grams of the dehydrated Martin-Lewis Agar in 500 ml of purified filtered water. Use a suitable container that can withstand the heat and agitation.
2. Heat the suspension while agitating it frequently to ensure even distribution of the agar. Boil the mixture for one minute, maintaining agitation throughout this step.
3. After boiling, sterilize the medium by subjecting it to autoclaving at 121ºC for 15 minutes. Autoclaving is a process that utilizes high-pressure steam to kill any microorganisms present in the medium.
4. Once the sterilization process is complete, allow the medium to cool to a temperature of 45-50ºC. It is important to cool the medium sufficiently to avoid damaging any heat-sensitive components.
5. While the medium is cooling, prepare the additional components that need to be added. Measure 500 ml of sterile 2% Hemoglobin, which serves as a source of the X factor (hemin). Additionally, measure 10 ml each of Bio-X Enrichment and V.C.A.T. These enrichments provide the V factor (nicotinamide adenine dinucleotide – NAD) and other growth factors necessary for the optimal growth of pathogenic Neisseria species.
6. Once the medium has reached the desired temperature, add the sterile 2% Hemoglobin, Bio-X Enrichment, and V.C.A.T. to the cooled agar. Gently mix the components to ensure uniform distribution.
7. Finally, dispense the prepared Martin-Lewis Agar into sterile Petri dishes or other suitable containers. Take care to maintain aseptic conditions during this step to prevent contamination.

The prepared Martin-Lewis Agar is now ready for use in the isolation and cultivation of pathogenic Neisseria species from clinical specimens containing mixed microbial flora. Proper storage and handling procedures should be followed to maintain the quality and sterility of the agar until it is used in the laboratory setting.

Performance Test Procedure

The performance test procedure for Martin-Lewis Agar involves inoculating representative samples with specific cultures and observing the growth and characteristics of the colonies. Here are the steps involved:

1. Inoculation: Take representative samples of the organisms you want to test. For Neisseria gonorrhoeae and N. meningitidis strains, add 0.1 milliliters of a dilution containing 30 to 300 colony-forming units (CFU) per 0.1 milliliters to each agar plate. For other organisms, use dilutions containing between 104 and 105 CFU/0.1 mL. Spread-inoculate the samples evenly on the agar plates using a sterile glass spreader.
2. Incubation: Place the inoculated plates in an incubator set at a temperature of 35 +/- 2 degrees Celsius. The incubation should be carried out under an aerobic atmosphere containing 35 percent carbon dioxide. This specific atmosphere is favorable for the growth of Neisseria species.
3. Control Plates: Include nonselective, selective, and non-selective controls in the test. Use Chocolate II-Agar plates for the nonselective control, which allows for the growth of a wide range of organisms. The selective control is not specified in the given information. These control plates will help evaluate the performance of the medium and ensure its suitability for the intended purpose.
4. Examination: After incubation, examine the plates at 18-24 hours and again at 48 hours. Observe the size and appearance of the colonies that have grown on the agar. Pay attention to the selective nature of the medium and look for the growth of the target organisms while inhibiting the growth of other unwanted microorganisms. Note any specific characteristics or features of the colonies.

Result of Martin Lewis Agar

The results obtained from Martin-Lewis Agar can provide information about the colonial morphology of specific microorganisms, particularly Neisseria gonorrhoeae and Neisseria meningitidis. Here are the typical results observed on Martin-Lewis Agar:

1. Neisseria gonorrhoeae: The colonies of Neisseria gonorrhoeae on Martin-Lewis Agar typically appear as small grayish-white to colorless mucoid colonies. These colonies may exhibit a smooth or slightly rough texture. The size of the colonies may vary, but they are generally smaller in comparison to other organisms present in the sample.
2. Neisseria meningitidis: On Martin-Lewis Agar, colonies of Neisseria meningitidis tend to be medium to large in size and display a distinctive blue-gray color. These mucoid colonies can be easily distinguished due to their characteristic coloration. They may appear moist or mucoid in texture.

It’s important to note that these descriptions represent the typical colonial morphology observed for Neisseria gonorrhoeae and Neisseria meningitidis on Martin-Lewis Agar. However, variations in colonial appearance can occur depending on factors such as strain diversity and growth conditions.

The specific colonial characteristics observed on Martin-Lewis Agar aid in the preliminary identification of Neisseria gonorrhoeae and Neisseria meningitidis, contributing to the diagnostic process for these pathogens. Additional confirmatory tests and techniques are usually required for a definitive identification.

Organism	Expected Results
Neisseria gonorrhoeae ATCC 43069	Growth
Staphylococcus epidermidis ATCC 12228	Inhibition
Proteus mirabilis ATCC 12453	Inhibition
Escherichia coli ATCC 25922	Inhibition
Candida albicans ATCC 10231	Inhibition

Storage Instructions

When you receive the plates, keep them in the dark, at 2-8 degC. Avoid overheating and freezing. Keep the lid closed until you are the product is ready for use. Reduce exposure to sunlight. Plates that have been prepared and kept in the original sleeve wrapper at temperatures between 2 and 8 degrees Celsius until the time to use can be inoculated until the expiration date, and incubated according to suggested incubation time. Let the medium cool to room temperature prior to inoculation. JEMBEC CO2 bags and tablets can be kept with the plates between 2 and 8 degC or separate with the plates room temperature.

Warnings and Precautions

For in vitro Diagnostic Use. If there is excessive moisture place the bottom on top of an off-set lid and let it air dry to avoid the formation of an airtight seal between the bottom and top of the dish during the incubation. Infectious microorganisms, like Hepatitis virus and Human Immunodeficiency Virus, may be detected in clinical samples. “Standard Precautions”20-23 and guidelines from the institution should be followed when handling all objects that have been contaminated with blood or various body fluids. After use, dishes, containers for specimens, and other materials that are contaminated must be sterilized with autoclaves before throwing them away.

Uses of Martin Lewis Agar

Martin-Lewis Agar has several uses in the field of microbiology and clinical diagnostics. Here are the primary applications of Martin-Lewis Agar:

1. Selective Isolation of Neisseria Species: Martin-Lewis Agar is commonly used as a selective medium for the isolation and cultivation of Neisseria species, including Neisseria gonorrhoeae and Neisseria meningitidis. The composition of the agar, including selective agents and enrichments, promotes the growth of these pathogenic Neisseria species while inhibiting the growth of unwanted microorganisms. It provides an environment conducive to the recovery and isolation of Neisseria strains from clinical specimens containing mixed microbial flora.
2. Recovery of Neisseria gonorrhoeae: Martin-Lewis Agar, when supplemented with lincomycin, provides a rich and selective growth medium specifically designed for the recovery of Neisseria gonorrhoeae. The addition of lincomycin reduces the concentration of vancomycin in the medium while still maintaining selective properties. This modified formulation is particularly effective for isolating Neisseria gonorrhoeae from both genital and oropharyngeal specimens, allowing for accurate diagnosis and appropriate treatment.
3. Diagnostic Applications: Martin-Lewis Agar plays a crucial role in the laboratory diagnosis of Neisseria infections, especially gonorrhea. It enables the growth and isolation of pathogenic Neisseria species, facilitating the identification of specific strains causing infections in patients. The selective nature of the medium helps differentiate Neisseria species from other commensal bacteria and mixed microbial populations commonly found in clinical samples.
4. Research and Epidemiological Studies: Martin-Lewis Agar is also utilized in research studies and epidemiological investigations focused on Neisseria species. It provides a reliable and standardized medium for the isolation, cultivation, and characterization of pathogenic Neisseria strains. The ability to selectively grow and identify these organisms aids in understanding their distribution, antibiotic susceptibility patterns, and genetic diversity.

Overall, Martin-Lewis Agar serves as an essential tool in the laboratory for the selective isolation and identification of pathogenic Neisseria species. It has significant applications in clinical diagnostics, research, and epidemiological studies related to Neisseria infections, particularly gonorrhea. The medium’s selective properties and modified formulations optimize the recovery of target organisms, leading to improved patient care and enhanced understanding of Neisseria-related diseases.

Limitations of Martin Lewis Agar

Martin-Lewis Agar, like any diagnostic medium, has certain limitations that should be taken into consideration. Here are the limitations associated with the use of Martin-Lewis Agar:

1. Confirmatory Tests Required: Although Martin-Lewis Agar provides selective growth conditions for Neisseria species, additional tests such as morphological, biochemical, and serological tests should be performed for final identification and confirmation of the findings. The agar serves as an initial screening medium, but further characterization is necessary for accurate identification.
2. Sensitivity of Neisseria gonorrhoeae: Neisseria gonorrhoeae is a fastidious organism that is sensitive to desiccation and temperature extremes. These characteristics can impact the ability to detect the microorganism through culture techniques. Factors such as improper specimen collection, storage, and inoculation, initiation of antimicrobial therapy before specimen collection, incorrect incubation temperatures and atmospheres, improper length of culture incubation, and mishandling of culture media can affect the growth and detection of N. gonorrhoeae.
3. Inhibition by Antimicrobial Agents: Some strains of N. gonorrhoeae may be inhibited by the presence of vancomycin or trimethoprim lactate, which are included in the selective agents of Martin-Lewis Agar. This inhibition can lead to false-negative results, where the growth of N. gonorrhoeae is suppressed, making it difficult to detect.
4. Similar Colonies from Other Organisms: Certain oxidase-positive, gram-negative bacilli may grow on Martin-Lewis Agar and produce colonies that resemble those of N. gonorrhoeae. This similarity in colony morphology can lead to misidentification or confusion during the interpretation of results. Therefore, confirmatory tests are necessary to differentiate these organisms from true N. gonorrhoeae strains.

It is important to be aware of these limitations when utilizing Martin-Lewis Agar for the isolation and identification of Neisseria species. Proper specimen collection, handling, and performing additional tests are crucial for accurate identification and reliable results.

Quality Control

Quality control measures are essential to ensure the reliability and performance of Martin-Lewis Agar. Here are the aspects of quality control for this medium, as indicated in the provided information:

1. Appearance: Martin-Lewis Agar should appear as a cream to yellow homogeneous free-flowing powder. Any deviations from this appearance could indicate potential issues with the quality or stability of the medium.
2. Gelling: The agar should form a firm gel comparable to a 1.3% agar gel. This property ensures proper solidification and consistency of the medium.
3. Colour and Clarity of Prepared Medium: After preparing the medium with the addition of hemoglobin or sterile lysed blood and supplements, it should exhibit a chocolate-colored opaque gel appearance in Petri plates. This indicates that the medium has been properly reconstituted and the desired characteristics have been achieved.
4. pH: The pH of a 4.2% w/v aqueous solution of the medium should be within the range of 6.80-7.20. This pH range ensures the optimal growth conditions for the target organisms and the functionality of the medium.
5. Cultural Response: The cultural response of the medium can be assessed by inoculating it with specific organisms and observing their growth and colony characteristics. The provided information includes the cultural response of Martin-Lewis Agar to various organisms after incubation at 35-37°C for 18-48 hours. The expected growth and colony appearance for each organism are indicated, and this serves as a reference for assessing the performance of the medium.

It is crucial to follow the recommended quality control procedures during the preparation and use of Martin-Lewis Agar to ensure accurate and reliable results. Regular monitoring and adherence to quality control measures help maintain the integrity and effectiveness of the medium for its intended applications.

FAQ

References

• https://www.bd.com/resource.aspx?idx=8980
• https://exodocientifica.com.br/_technical-data/M413.pdf
• https://www.himedialabs.com/in/m2085-martin-lewis-agar.html
• https://hardydiagnostics.com/e39
• https://www.dalynn.com/dyn/ck_assets/files/tech/PC60.pdf
• https://www.keyscientific.com/product.php?productid=303
• https://www.cdhfinechemical.com/martin-lewis-agar-plate