Martin Lewis Agar, a specialized culture medium, is a valuable tool in microbiology and clinical laboratories. It is particularly effective in isolating and identifying pathogenic Neisseria species, such as Neisseria gonorrhoeae and Neisseria meningitidis. These organisms are the causative agents of serious infections like gonorrhea and meningitis, which require prompt diagnosis and treatment. The use of Martin Lewis Agar significantly aids in this process, contributing to the effective management of these infections.
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Historical Context
Developed as an enhancement of Thayer-Martin Agar, Martin Lewis Agar incorporates a similar principle of selective inhibition. Thayer-Martin Agar was initially created to address the challenge of isolating Neisseria gonorrhoeae amidst various contaminating microorganisms from clinical specimens. Martin Lewis Agar further optimizes this process by adjusting the antibiotic composition and concentrations to improve the selectivity and recovery rates of the target pathogens.
Principle of Martin Lewis Agar
Martin Lewis Agar’s principle is based on its use as a selective medium for isolating pathogenic Neisseria species, particularly Neisseria gonorrhoeae and Neisseria meningitidis. It is a modified version of Thayer-Martin agar and incorporates several key ingredients to suppress the growth of contaminating organisms while promoting the development of Neisseria species.
Working Principle:
The agar medium works by creating an environment that selectively allows the growth of Neisseria species while inhibiting other bacteria and fungi. This is achieved through:
- Selective Inhibition: The antibiotics incorporated in the medium are carefully chosen to suppress the growth of contaminants without affecting the target Neisseria species. For example, vancomycin inhibits Gram-positive bacteria, colistin inhibits Gram-negative bacteria other than Neisseria, nystatin prevents fungal growth, and trimethoprim inhibits Proteus species.
- Nutrient-Rich Environment: The base medium, supplemented with hemoglobin and other growth factors, ensures that Neisseria species have the necessary nutrients to grow.
- Supportive Additives: Supplements like IsoVitaleX provide additional growth factors essential for Neisseria’s growth.
Interpretation:
- Positive Result: The presence of typical Neisseria colonies can be confirmed using biochemical tests, Gram staining, or molecular methods.
- Antagonistic Result: The absence of typical colonies indicates that Neisseria species were not present in the sample or below detectable levels.
Composition of Martin Lewis Agar
Martin Lewis agar is modified Thayer Martin agar with an increased level of vancomycin. Its base is chocolate agar. Its final pH is 7.2 ± 0.22 at 25°C. The antibiotics in the agar include vancomycin, colistin, anisomycin, and trimethoprim. It also comprises a supplement solution with L-cysteine HCI, glucose, L-glutamine, NAD (nicotinamide adenine diphosphate), and other components.
Composition per liter:
| Components | Amount |
| Pancreatic digest of casein | 7.5 g |
| Hemoglobin (Hb) | 10.0 g |
| Selected meat peptone | 7.5 g |
| NaCl (sodium chloride) | 5 g |
| K2HPO4 (Dipotassium phosphate) | 4 g |
| Corn starch | 1 g |
| KH2PO4 (Monopotassium phosphate) | 1 g |
| Supplement solution | 10 ml |
| VCAT inhibitor | 10 ml |
| Agar | 12 g |
| Final pH 7.2 ± 0.22 at 25°C |
Source: Martin-Lewis agar is available as a prepared medium from BD Diagnostic Systems.
Composition of Supplement Solution per liter:
| Components | Amount |
| Glucose | 100 g |
| L-cysteine HCI | 25.9 g |
| L-glutamine | 10 g |
| L-cystine | 1.1 g |
| Adenine | 1 g |
| Nicotinamide adenine dinucleotide | 0.25 g |
| Vitamin B12 | 0.1 g |
| Thiamine pyrophosphate | 0.1 g |
| Guanine HCl | 0.03 g |
| Fe(NO3).6H2O | 0.02 g |
| p-Aminobenzoic acid | 0.013 g |
| Thiamine HCl | 3.0 mg |
Source: The supplement solution IsoVitaleX® enrichment is available from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems.
Composition of VCAT inhibitor per 10.0mL
| Components | Amount |
| Colistin | 7.5 mg |
| Trimethoprim lactate | 5 mg |
| Vancomycin | 4 mg |
| Anisomycin | 0.02 g |
Preparation of Martin Lewis Agar
- Add the components, except supplement solution and VCAT inhibitor, to distilled/deionized water
- Then, bring the volume to 980.0mL.
- After that gently heat while stirring and bring to boiling.
- Now, autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
- Then, aseptically add sterile supplement solution and sterile VCAT inhibitor. Mix thoroughly.
- Finally, pour into sterile Petri dishes.
Storage and Shelf Life
Dehydrated media is stored in the dark in a sealed container below 30°C. However, the prepared media should be stored under refrigeration (2-8°C) and used within 60 days of preparation.
Using media that shows signs of deterioration (such as shrinking, cracking, or discoloration) or contamination, or has exceeded its expiration date, can lead to inaccurate results and compromised experiments. Hence, it’s important to avoid such media.
Uses of Martin Lewis Agar
- The increased level of vancomycin inhibits the growth of Gram-positive bacteria. Colistin also inhibits the growth of Gram-negative bacteria, Anisomycin suppresses the growth of fungi, and Trimethoprim inhibits the growth of Proteus spp. So, it is used for the isolation and cultivation of pathogenic Neisseria from specimens containing mixed flora of bacteria and fungi.
- Enrichment of Martin Lewis agar by the addition of Penicillin G aids in recovering pathogenic Neisseria species, especially penicillinase-producing species, from mixed flora.
Limitation of Martin Lewis Agar
1. Due to nutritional variations, certain strains may show poor growth.
2. Antibiotics may inhibit certain strains of Neisseria gonorrhea.
3. An enriched non-selective medium must be used in parallel.
4. Further biochemical and serological tests must be carried out for confirmation
Refernces
- Martin, J. E., Armstrong, J. H., & Smith, P. B. (1974). New system for cultivation of Neisseria gonorrhoeae. Applied microbiology, 27(4), 802–805. https://doi.org/10.1128/am.27.4.802-805.1974
- Granato, P. A., Paepke, J. L., & Weiner, L. B. (1980). Comparison of modified New York City medium with Martin-Lewis Medium for recovery of Neisseria gonorrhoeae from clinical specimens. Journal of clinical microbiology, 12(6), 748–752. https://doi.org/10.1128/jcm.12.6.748-752.1980
- Atlas, R.M., & Snyder, J.W. (2013). Handbook of Media for Clinical and Public Health Microbiology (1st ed.). CRC Press. https://doi.org/10.1201/b15973
- Thayer J. and Martin J.E. Jr., 1966, Public Health Rep., 81:559