Culture Media

Martin Lewis Agar Composition, Preparation, Principle, Uses

Thayer-Martin Agar was created in 1964 to identify of N. Gonorrhoeae as well as N. meningitidis. Another modification to these formulations was...

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This article writter by MN Editors on December 09, 2021

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

Thayer-Martin Agar was created in 1964 to identify of N. Gonorrhoeae as well as N. meningitidis. Another modification to these formulations was devised to enhance the separation of Neisseria sp. pathogenic. from samples, which had huge quantities of mixed microbial flora.

Martin-Lewis Agar is the formula that was developed after modification , which has more vancomycin, which is a stronger inhibitor of Gram-positive organisms. The creation of Martin-Lewis (ML) media is an improvement made more recently in the Thayer-Martin formulation that has led to greater recovery of gonococci in clinical samples. Martin Lewis Agar provides a selectivist and rich medium that is suitable to isolate and cultivate of Neisseria species isolated from mixed flora. Hemoglobin Bio-X, hemoglobin and dextrose that are included in the culture media supply enough nutrients to allow for a rapid growth of microorganisms that are fastidious.

Composition of Martin Lewis Agar

IngredientsGms/L
Casein Peptone7.5
Meat Peptone7.5
Corn Starch1.0
Potassium Phosphate, dibasic4.0
Potassium Phosphate, monobasic1.0
Sodium Chloride5.0
Agar10.0
Hemoglobin10.0
Dextrose1.5
Enrichment Bio-X10 ml
Antibiotics V.C.A.T10 ml

pH 7.2 +/- 0.2 at 25º C

Enrichment Bio-X

Purified Water Vitamin B120.01 g
Thiamine Pyrophosphate0.1 g
L-Glutamine10.0 g
Ferric Nitrate0.02 g
Adenine1.0 g
Thiamine Hydrochloride0.003 g
Guanine Hydrochloride0.03 g
L-Cysteine Hydrochloride25.9 g
p-Aminobenzoic Acid0.013 g
L Cystine1.1 g
Nicotinamide Adenine Dinucleotide0.25 g
Dextrose100.0 g

Preparation of Martin Lewis Agar

  1. Subsist 36 g of media that has been dehydrated in 500ml of water purified and filtered.
  2. Stir frequently and heat to boil for 1 minute.
  3. Sterilize at 121oC for 15 minutes.
  4. Cool to 45-50o C.
  5. Add 500 milliliters of pure 2 % Hemoglobin 10 ml of Bio-X Enrichment , and 10ml of V.C.A.T.
  6. Mix thoroughly and pour into sterilized Petri dishes.

Performance Test Procedure

  1. Innoculate representative samples using the following cultures.
    1. To test for the Neisseria gonorrhoeae as well as N. meningitidis strains add 0.1 milliliters of a dilution with 30 to 300 CFU/0.1 milliliters to each dish and spread-inoculate with a sterile glass spreader. For other organisms, utilize dilutions with between 104 and 105 CFU/0.1 mL . Spread-inoculate the samples using the spreader of sterile glass.
    2. Incubate the plates for 35 +/- 2degC under an atmosphere that is aerobic and contains 35 percent carbon dioxide.
    3. Use Chocolate II-Agar plates for nonselective, nonselective and non-selective controls.
  2. Examine the plates after 18-24 and 48 hours for the size of colonies, growth and selection.
  3. Expected Results

Principle of Martin Lewis Agar

Martin Lewis Agar consists of the chocolate agar base that has an enhanced GC Agar base bovine hemoglobin and an enrichment chemically defined. The GC base is a source of nitrogenous nutrients in the form of meat and casein peptones and phosphate buffer to keep pH and cornstarch to neutralize toxic fatty acids which could be found within the agar. Hemoglobin is a the X factors (hemin). Chemically-defined enrichments, BioX enrichment, offer V factor (nicotinamide Adenine Dinucleotide NAD) as well as vitamins amino acids, coenzymes ferric ions, dextrose as well as other growth factors that are essential to promote growth and improvement of the pathogenic Neisseria species. The specific medium consists vancomycin, an antimicrobial agent colistin, anisomycin, colistin (V-C-A inhibitor) and trimethoprim to inhibit the normal bacteria. Vancomycin is active against Gram-positive bacteria. Colistin blocks gram-negative bacteria which includes Pseudomonas species. Anisomycin blocks yeasts, and Trimethoprim hinders Proteus.

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.

Result of Martin Lewis Agar

The typical colonial morphology of Martin-Lewis Agar

  • Neisseria gonorrhoeae:  Small, grayish-white to colorless, mucoid colonial colonies.
  • Neisseria meningitidis:  Medium-sized to large blue-gray mucoid colonies.
Result of Martin Lewis Agar
Result of Martin Lewis Agar
OrganismExpected 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 is an enriched media to isolate selectively Neisseria species.
  • Martin Lewis with Lincomycin provides the most potent, specific growth medium with reduced vancomycin levels for the removal of Neisseria gonorrhoeae in both oropharyngeal and genital specimens.

Limitations of Martin Lewis Agar

  • Further tests such as biochemical, morphological and/or serological tests are recommended to verify the results.
  • N. gonorrhoeae, which are sensitive organisms that are prone to temperature extremes and desiccation. The capability to identify microorganisms through culture techniques may be affected by these factors: incorrect specimen collection as well as storage and inoculation beginning anti-infective therapies prior to collection of specimens as well as improper temperature for incubation and atmospheres, incorrect duration of the incubation period and the improper use of material, the handling and storage of storage of culture media and handling of culture media and handling.
  • Certain varieties of N. Gonorrhoeae could be inhibited by vancomycin as well as some are inhibited by trimethoprim lactate.
  • Certain oxidase positive, gram-negative bacteria will thrive on selective media, and will produce colonies that resemble N. Gonorrhoeae.

Quality Control

  • Examine the plates in accordance with “Product Deterioration.”
  • Take a look at the plates in a visual way to ensure that any physical defects won’t affect the use.
  • Find the pH potentiometrically at ambient temperature, to ensure compliance to the specifications of 7.2 + 0.2.
  • Take note of the firmness of the plates during the process of inoculation.
  • Incubate the uninoculated plates at 35 + – 2degC for 72 hours before examining for contamination by microbial species.
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