Martin Lewis Agar- Composition, Principle, Preparation, Results, Uses

Martin-Lewis Agar is a type of chocolate agar that is used for the selective isolation and cultivation of pathogenic Neisseria species, such as Neisseria gonorrhoeae and Neisseria meningitidis. These bacteria are the causative agents of gonorrhea and meningitis, respectively, and are important public health concerns worldwide. Martin-Lewis Agar was developed by Martin and Lewis in 1980 as an improvement over Thayer-Martin Agar, which was the standard medium for Neisseria isolation at that time. Martin-Lewis Agar has a higher recovery rate and a lower contamination rate than Thayer-Martin Agar, making it more suitable for clinical and laboratory use. Martin-Lewis Agar contains a chocolate agar base that is enriched with hemoglobin and Bio-X, a chemically defined supplement that provides essential growth factors for Neisseria. It also contains four antimicrobial agents: vancomycin, colistin, anisomycin, and trimethoprim, which inhibit the growth of most other bacteria and fungi that may be present in the specimens. Martin-Lewis Agar is a differential medium that allows the identification of Neisseria species based on their colony morphology, oxidase reaction, and carbohydrate utilization tests. In this article, we will discuss the composition, principle, preparation, results, uses, and limitations of Martin-Lewis Agar.

Martin Lewis Agar is a modified chocolate agar that contains several ingredients to enhance the growth and isolation of Neisseria species. The main components of Martin Lewis Agar are:

• GC Agar base: This is a nutrient-rich base that provides casein and meat peptones as sources of nitrogen, phosphate buffer to maintain pH, and cornstarch to neutralize toxic fatty acids that may be present in the agar.

• Bovine hemoglobin: This is a source of X factor (hemin), which is required by some Neisseria species for growth and metabolism.

• Bio-X enrichment: This is a chemically defined supplement that provides V factor (nicotinamide adenine dinucleotide - NAD), vitamins, amino acids, coenzymes, dextrose, ferric ions, and other growth factors necessary for optimal growth of pathogenic Neisseria species.

• V-C-A Inhibitor: This is a selective agent that contains vancomycin, colistin, anisomycin, and trimethoprim. These antibiotics inhibit the growth of most gram-positive bacteria, gram-negative bacteria (except Neisseria), yeasts, and Proteus species, respectively. This allows for the selective isolation of Neisseria species from mixed flora specimens.

The final pH of Martin Lewis Agar is 7.2 +/- 0.2 at 25º C. The medium appears dark brown to black in color due to the presence of hemoglobin. The medium should be stored at 2-8º C and used within the expiration date indicated on the label. The medium should not be used if there are signs of deterioration, such as discoloration, contamination, or desiccation.

Martin Lewis Agar is a selective and differential medium for the isolation and identification of pathogenic Neisseria species, such as N. gonorrhea and N. meningitidis. These bacteria are gram-negative diplococci that require enriched media with specific growth factors and inhibitors to suppress the normal flora.

Martin Lewis Agar consists of a chocolate agar base, which is a heated blood agar that releases hemoglobin and other nutrients from the red blood cells. The chocolate agar base contains an improved GC Agar base, bovine hemoglobin, and a chemically defined enrichment. The GC base provides nitrogenous nutrients in the form of casein and meat peptones, phosphate buffer to maintain pH, and cornstarch to neutralize toxic fatty acids that may be present in the agar. Hemoglobin provides X factor (hemin), which is essential for the growth of N. gonorrhoeae and N. meningitidis. Chemically defined enrichments, Bio-X enrichment, provide V factor (nicotinamide adenine dinucleotide – NAD), vitamins, amino acids, coenzymes, dextrose, ferric ions, and other growth factors necessary for improvement and optimal growth of pathogenic Neisseria species.

The selective medium also comprises the antimicrobial agent`s vancomycin, colistin, anisomycin (V-C-A Inhibitor), and trimethoprim to suppress the normal flora. Vancomycin is active primarily against gram-positive bacteria. Colistin inhibits gram-negative bacteria, including Pseudomonas species. Anisomycin inhibits yeasts, and trimethoprim inhibits Proteus. These agents allow for the selective isolation of Neisseria species from specimens that may contain mixed flora.

The medium is also differential because it allows for the differentiation of N. gonorrhoeae and N. meningitidis based on their colony morphology and biochemical reactions. N. gonorrhea produces small grayish-white to colorless mucoid colonies, while N. meningitides produce medium to large, blue-gray mucoid colonies. Both species are oxidase-positive and catalase-positive, but they can be further distinguished by carbohydrate utilization tests. N. gonorrhea can utilize glucose but not maltose or sucrose, while N. meningitidis can utilize glucose and maltose but not sucrose.

Martin Lewis Agar is a useful medium for the selective isolation and identification of pathogenic Neisseria species from clinical specimens such as genital swabs, throat swabs, cerebrospinal fluid (CSF), blood cultures, etc.

Martin Lewis Agar is a modified chocolate agar that contains selective agents to inhibit the growth of normal flora and enhance the isolation of Neisseria species. To prepare Martin Lewis Agar, the following steps are required:

• Suspend 36 g of dehydrated media in 500 ml of purified filtered water. The dehydrated media contains GC Agar base, cornstarch, and agar.

• Heat the mixture with frequent agitation and boil for one minute to dissolve the media completely.

• Sterilize the mixture at 121º C for 15 minutes in an autoclave. This will ensure the elimination of any contaminating microorganisms.

• Cool the mixture to 45-50º C in a water bath or an incubator. This will prevent the denaturation of the heat-sensitive ingredients that will be added later.

• Add 500 ml of sterile 2% Hemoglobin, 10 ml of Bio-X Enrichment and 10 ml of V.C.A.T. The hemoglobin provides an X factor (hemin) for the growth of Neisseria species. The Bio-X Enrichment provides V factor (NAD), vitamins, amino acids, coenzymes, dextrose, ferric ions, and other growth factors for the optimal growth of pathogenic Neisseria species. The VCAT contains vancomycin, colistin, anisomycin, and trimethoprim as selective agents to inhibit normal flora.

• Mix gently and dispense into sterile Petri dishes. The final pH of the medium should be 7.2 +/- 0.2 at 25º C.

• Allow the medium to solidify and store at 2-8º C in a sealed plastic bag or container until use. The medium should be used within one month of preparation.

The prepared medium should be checked for sterility, color, clarity, and pH before use. The medium should also be warmed to room temperature before inoculation. The inoculated plates should be incubated at 35-37º C in a humidified atmosphere containing 5% CO2 for 24-48 hours. The growth and morphology of the colonies should be observed and recorded.

Martin Lewis Agar allows the growth of Neisseria species while inhibiting most of the normal flora. The colonies of Neisseria gonorrhoeae and Neisseria meningitides can be distinguished by their size, color, and morphology on this medium.

• Neisseria gonorrhoeae: Small grayish-white to colorless mucoid colonies. They are usually less than 1 mm in diameter and may be difficult to see without a magnifying lens.

• Neisseria meningitides: Medium to large, blue-gray, mucoid colonies. They are usually 1-2 mm in diameter and have a smooth, convex surface.

Other Neisseria species may also grow on Martin Lewis Agar, but they are usually less pathogenic and less common than N. gonorrhoeae and N. meningitidis. Some examples are:

• Neisseria lactamica: Medium to large, grayish-white, non-mucoid colonies. They are usually 2-3 mm in diameter and have a rough, irregular surface.

• Neisseria sicca: Small to medium, white to yellowish, non-mucoid colonies. They are usually 1-2 mm in diameter and have a smooth, flat surface.

• Neisseria subflava: Small to medium, white to yellowish, non-mucoid colonies. They are usually 1-2 mm in diameter and have a smooth, convex surface.

Some oxidase-positive gram-negative bacilli may also grow on Martin Lewis Agar and produce colonies resembling Neisseria species. These include Moraxella catarrhalis, Acinetobacter species, and Pseudomonas aeruginosa. Therefore, additional tests such as Gram stain, oxidase test, carbohydrate utilization test, and serological test should be performed to confirm the identification of Neisseria species.

Here is an example of what the results of Martin Lewis Agar may look like:

| Organism | Colony Morphology |

| --- | --- |

| Neisseria gonorrhoeae | Small grayish-white to colorless, mucoid colonies |

| Neisseria meningitides | Medium to large, blue-gray, mucoid colonies |

| Neisseria lactamica | medium to large, grayish-white, non-mucoid colonies |

| Neisseria sicca | Small to medium, white to yellowish, non-mucoid colonies |

| Neisseria subflava | Small to medium, white to yellowish, non-mucoid colonies |

| Moraxella catarrhalis | Small white to grayish-white, non-mucoid colonies |

| Acinetobacter species | Small white to pinkish-white, non-mucoid colonies |

| Pseudomonas aeruginosa | Small greenish-blue, non-mucoid colonies |

Martin Lewis Agar is an enriched medium for the selective isolation of Neisseria species, especially Neisseria gonorrhoeae and Neisseria meningitidis. These are the causative agents of gonorrhea and meningitis, respectively, which are serious infections that can lead to complications such as infertility, pelvic inflammatory disease, septicemia, arthritis, and brain damage. Therefore, it is important to diagnose and treat these infections promptly and accurately.

Martin Lewis Agar provides a suitable environment for the growth of Neisseria species by supplying them with essential nutrients and factors such as hemoglobin (X factor), Bio-X enrichment (V factor), and iron. It also inhibits the growth of most other bacteria that may be present in the clinical specimens by incorporating selective agents such as vancomycin, colistin, anisomycin, and trimethoprim. These agents target different groups of bacteria based on their cell wall structure, membrane permeability, protein synthesis, and folate metabolism.

Martin Lewis Agar can be used to isolate Neisseria species from various types of specimens, such as genital swabs, urethral swabs, cervical swabs, rectal swabs, pharyngeal swabs, blood, cerebrospinal fluid (CSF), and joint fluid. The specimens should be inoculated onto the medium as soon as possible after collection and incubated at 35-37°C in a humidified atmosphere containing 5-10% CO2 for 24-48 hours. The colonies of Neisseria species can be identified by their morphology, color, oxidase reaction, and carbohydrate utilization tests.

Martin Lewis Agar can also be modified by adding or reducing certain components to enhance its performance for specific purposes. For example, Martin Lewis Agar with Lincomycin is a variant that contains a lower concentration of vancomycin and an additional antibiotic, lincomycin. This modification improves the recovery of Neisseria gonorrhoeae from oropharyngeal specimens, which may contain other vancomycin-resistant bacteria that can interfere with the isolation. Another example is Martin Lewis Agar with Isovitalex Enrichment, which contains a different chemically defined supplement that provides more vitamins and cofactors for the growth of Neisseria species.

Martin Lewis Agar is a valuable tool for the clinical microbiology laboratory to isolate and identify Neisseria species from various specimens. It helps in the diagnosis and management of gonorrhea and meningitis infections, which can have serious consequences if left untreated. It also helps in the epidemiological surveillance and control of these infections by providing information on the prevalence and distribution of Neisseria strains in different regions and populations.

• Martin Lewis Agar is a selective medium that can isolate Neisseria species from other normal flora, but it cannot differentiate between different Neisseria species. Therefore, additional testing such as morphological, biochemical, and/or serological tests should be performed for final identification and confirmation of the findings.

• Neisseria gonorrhoeae and Neisseria meningitides are fastidious organisms that require optimal conditions for growth and survival. They are sensitive to desiccation and temperature extremes and can be easily killed by exposure to air or light. The ability to detect these microorganisms by culture techniques can be affected by several factors, such as:

• Improper specimen collection, storage, and inoculation: Specimens should be collected using sterile swabs and transported in appropriate transport media (such as Amies or Stuart) as soon as possible. Specimens should be inoculated onto the medium within 2 hours of collection and incubated at 35-37°C in a humidified atmosphere with 5-10% CO2.

• Initiation of anti-infective therapy prior to specimen collection: Antibiotics can inhibit the growth of Neisseria species and reduce the sensitivity of culture methods. Therefore, specimens should be collected before the initiation of antibiotic therapy or as soon as possible after the therapy has started.

• Improper culture incubation temperatures and atmospheres: The optimal temperature for Neisseria species is 35-37°C, and the optimal atmosphere is a humidified environment with 5-10% CO2. Deviations from these conditions can affect the growth and viability of the organisms.

• Improper length of culture incubation: The recommended incubation time for Neisseria species is 18-24 hours. Longer incubation times can result in overgrowth of normal flora or other contaminants, while shorter incubation times can result in false-negative results due to insufficient growth.

• Improper material, storage, and handling of culture media: The culture media should be prepared according to the manufacturer`s instructions and stored at 2-8°C until use. The media should be brought to room temperature before inoculation and checked for signs of deterioration or contamination. The media should not be used beyond the expiration date or if there is any evidence of discoloration, turbidity, cracking, or drying.

• Some strains of N. gonorrhea may be inhibited by vancomycin and some by trimethoprim lactate, which are components of the selective agents in Martin Lewis Agar. These strains may produce smaller or fewer colonies than expected or may not grow at all on the medium.

• Certain oxidase-positive, gram-negative bacilli, such as Moraxella catarrhalis, Acinetobacter spp., Pseudomonas spp., Aeromonas spp., and Vibrio spp., can grow on selective media and produce colonies resembling N. gonorrhea or N. meningitidis. These organisms can be differentiated from Neisseria species by their morphology, Gram stain reaction, oxidase test results, carbohydrate utilization tests, and other biochemical tests.

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