TECHNICAL DATA SHEET #230 REV. 4

CHOCOLATE AGAR MEDIA (GC MEDIA BASE) SELECTIVE AND NONSELECTIVE PRODUCTS: Plated, Tubed, and Bottled Media:a Chocolate Agar with Enrichment GC Media without Hemoglobin Martin-Lewis Media (MLM) Modified Martin-Lewis Media (MMLM) Modified Thayer-Martin (MTM) Modified, Modified Thayer-Martin Agar (MMTM) Thayer-Martin Vancomycin Agar (TMV) Chocolate/Modified Martin-Lewis Media (MMLM) Chocolate/Modified Thayer-Martin (MTM) Chocolate/Martin-Lewis Media (MLM) a

P1250, T6280, T6281 P1261, P3104 (150 mm) P1953 P1950, P3600 (JEMBEC), P3602 (Pill-Pocket) 303444, P3594 (JEMBEC) P1960 P2455 P4100 (bi-plate) P4110 (bi-plate) P4800 (bi-pocket)

see catalog for ordering options

PURPOSE: Chocolate Agar media are used for the isolation and cultivation of fastidious microorganisms, primarily pathogenic Neisseria and Haemophilus species.

PRINCIPLE: Pathogenic Neisseria and Haemophilus species, among other fastidious microbes, will grow only in the presence of certain nutrients which can be found in Chocolate Agar. Originally, red blood cells were added to a melted nutrient base and heated to approximately 85°C which lysed the red blood cells, releasing into the base media hemoglobin and other nutrients present in lysed blood.9 Now these ingredients, hemoglobin and XV Factor Enrichment, are supplements added to the nutrient GC Base media. XV Factor Enrichment is rich in vitamins, additional cofactors, and amino acids. GC Media base consists of a complementary mixture of animal and vegetable peptones, cornstarch, and salts and was discovered as a result of the studies done by Johnston13 in 1945. Scientists, including Thayer, Martin, and Lester,1,6-8,10,11,12,15,16 further modified the Chocolate Agar formula by adding antibiotics and increasing the dextrose and agar concentrations, resulting in selective media used for the isolation of pathogenic members of the family Neisseriaceae from clinical specimens with large numbers of mixed normal flora. Vancomycin inhibits gram-positive bacteria; colistin inhibits gram-negative bacteria; nystatin inhibits some yeasts; and trimethoprim inhibits the growth of swarming Proteus species. Modifications continued to evolve due to discoveries found in the clinical setting; some strains of Candida albicans inhibit the growth of some N. gonorrhoeae and nystatin proved ineffective in inhibiting C. albicans.4,14 The antifungal agent, anisomycin, was substituted for nystatin and has proved effective (Martin-Lewis Media). The concentration of vancomycin was increased from 3 mcg/ml to 4 mcg/ml for greater inhibition of gram-positive bacteria6 but the discovery of a significant number of N. gonorrhoeae being susceptible to low concentrations of vancomycin has lead to lowering the concentration back to 2 mcg/ml (Modified MartinLewis Media). Modifications continued with the replacement of anisomycin with amphotericin B, and the addition of lincomycin to the formula. In addition to discovering media that would assist in the effective recovery of pathogenic Neisseriaceae, transport systems were developed to improve the recovery of gonococci which are very susceptible to adverse environmental conditions, to delays in transport, and which may be present in clinical specimens only in small numbers. The JEMBEC* (John E. Martin Biological Environmental Chamber) and the Pill-Pocket Systems, consisting of selective media, CO2-generating tablets, and small gas-impermeable plastic bags also aid in the recovery of gonococci. The chambers contain an inner well molded into the plate in which a CO2-generating tablet (sodium bicarbonate and citric acid) is inserted. Moisture activates the tablet which then provides a CO2 atmosphere conducive for the growth of gonococci. Note: Modified, Modified Thayer-Martin Media (MMTM) and Martin Lewis Media (MLM) have the same formula and will demonstrate

27120 SW 95th Avenue • Wilsonville, OR 97070 • 800.547.0659 Page 1

TECHNICAL DATA SHEET #230 REV. 4 identical performance. *registered trademark of Miles Scientific

FORMULAS: Approximate, per liter of deionized filtered water. (1)

Chocolate Agar with Enrichment: Peptic Digest of Animal Tissue ............ 7.5 g Pancreatic Digest of Casein ................ 7.5 Cornstarch........................................... 1.0 Dipotassium Phosphate ...................... 4.0 Sodium Chloride.................................. 5.0 Agar.................................................... 14.5 Hemoglobin ........................................ 10.0 XV Factor Enrichment ........................ 10.0 ml Final pH 7.2 ± 0.2 at 25°C

(2)

GC Media without Hemoglobin: Same as (1) above without hemoglobin Final pH 7.3 ± 0.1 at 25°C

(3)

Selective Media: Same as (1) above with the addition of 1.5 g of dextrose and:

Vancomycin MLM, MMTM MMLM MTM

4.0 mg 2.0 4.0 (4)

Amphotericin Colistin 2.0 mg 2.0 -

Trimethoprim Nystatin

7.5 mg 7.5 13.6

5.0 mg 5.0 5.0

Lincomycin

1.0 mg 1.0 0.0125 mu -

Agar -

Thayer Martin Vancomycin: Same as (1) above with the addition of 12.5 ml TMV Enrichment and 10.0 ml of TMV Supplement

PRECAUTIONS:* For in vitro diagnostic use. Observe approved biohazard precautions. Storage: Upon receipt store at 2-8°C away from direct light. Media should not be used if there are signs of contamination, deterioration (shrinking, cracking, or discoloration), or if the expiration date has passed. Limitations: Some strains of gonococci, usually arginine, hypoxanthine, uracil, (AHU) strains, are inhibited by vancomycin, and other gonococci are inhibited by trimethoprim. These strains can grow on nonselective Chocolate Agar media. See “New York City Media, Modified,” as an additional option for a selective media. Normal flora of the pharynx, female genital tract, and the rectum grow more rapidly than gonococci and can inhibit gonococcal growth. Certain gram-positive cocci will grow through on selective media incubated in excess of 48 hours. Chocolate Agar with Enrichment will not support the growth of all fastidious organisms; some organisms require further enrichment in order to be recovered from clinical specimens.

PROCEDURE:* Specimen Collection: Temperature and CO2 requirements of some fastidious organisms require that the processing of specimens be rapid; plated specimens for the recovery of gonococci and meningococci should be incubated immediately upon collection. Information on specimen collection is found in standard reference material. In general, specimens should be protected from extreme heat, cold, or desiccation and delivered to the microbiology laboratory without delay. If delay is unavoidable, use of a buffered holding media such as Amies Charcoal has proven effective for the recovery of most microorganisms. The recovery of gonococci, because they are very susceptible to adverse environmental conditions, has been more effective with the use of biological environment chambers inoculated directly at the patient care area.

27120 SW 95th Avenue • Wilsonville, OR 97070 • 800.547.0659 Page 2

TECHNICAL DATA SHEET #230 REV. 4 Method of Use, Chocolate Agar Media: Prior to inoculation, the media should be brought to room temperature. Inoculate the media by rolling a swab onto the agar, making certain all areas of the swab touch the agar, or by dropping 1-2 drops of a liquid specimen the size of a 25 cent piece onto the media. Using standard microbiological procedures, streak the inoculum so as to obtain isolated colonies. Incubate at 35°C in 5-10% CO2 for 18-72 hours. Examine daily for growth of possible pathogens. Perform Gram stains and biochemical and/or serological tests to identify definitively the microbes cultured. See appropriate references for further details. Method of Use, Selective Chocolate Agar Media: Prior to inoculation, the media should be brought to room temperature. Heavily inoculate the selective media by rolling the swab across the plate, forming a large “Z” pattern. Streak the plate in order to obtain isolated colonies. Incubate as above. Method of Use, Transport Systems: Biological Environment Chambers (JEMBEC, Pill-Pocket): After inoculation, place a CO2-generating tablet in the molded well. Replace the lid and place the chamber into the gas-impermeable plastic bag and securely close the bag. Allow the moisture from the media to activate the tablet and invert the plate after the pill is moistened. For maximum recovery using nutritive transport systems, incubate at 35°C for 18-24 hours before delivering to the laboratory. Upon arrival at the microbiology laboratory, examine the transport plates for growth; continue to incubate at 35°C in 5-10% CO2 for a total of 72 hours, as is done for routine selective media. Examine daily for morphologically typical colonies and presumptively or definitively identify microbial growth with further biochemical and/or serological tests. See appropriate references for further details. Interpretation: Chocolate Agar media, when properly inoculated with a specimen, will exhibit isolated colonies. N. gonorrhoeae grows as a small, mucoid colony, colorless to grayish-white, while N. meningitidis usually forms a larger, bluish-gray mucoid colony. In both cases the colonies are raised, finely granular, glistening, and convex. A Gram stain should be performed for any colonies appearing to be Neisseria and tested for oxidase production. Testing for carbohydrate utilization and/or serological tests need also to be performed. Haemophilus species are strict parasites, growing only in the presence of certain growth factors, X (hemin, hemoglobin) or V (coenzyme 1, NAD), or both. Microscopic examination shows small, usually pleomorphic, gram-negative rods, coccobacilli, or threadlike organisms. Haemophilus influenzae, the major pathogen of this genus, requires both X and V factors. Growth appears on Chocolate Agar in 18-24 hours as small, colorless, transparent, moist colonies with a distinct “mousey” odor. A Gram stain should be performed for any colonies appearing to be Haemophilus, and examined for hemolytic reactions, and biochemically and/or serologically tested. Material Required but Not Provided: Standard microbiological supplies and equipment commonly found in a microbiological laboratory are not provided.

QUALITY CONTROL:* Media Used: Chocolate Agar with Enrichment

Microorganisms Used (ATCC #): Neisseria gonorrhoeae (43069) Haemophilus influenzae (10211) Streptococcus pneumoniae (6305)

Expected Results: Growth Growth Growth

GC Media without Hemoglobin

Neisseria gonorrhoeae (49226)

Growth

MTM, MMTM, TMV, MLM, MMLM

Neisseria gonorrhoeae (43070, 49226, 43069) Neisseria meningitidis (13090) Candida albicans (60193) Neisseria sicca (9913) Staphylococcus epidermidis (12228) Escherichia coli (25922) Proteus mirabilis (43071)

Growth Growth Growth; Inhibition (MMLM, MMTM) Inhibition, partial to complete Inhibition, partial to complete Inhibition, partial to complete Inhibition, partial to complete

User Quality Control: Check for signs of contamination and deterioration and test the ability of the Chocolate Agar media, selective and nonselective, to support the growth of Neisseria gonorrhoeae as defined by the regulatory agencies. Chocolate Agar media should appear firm, opaque, and chocolate brown in color with the exception of GC Media without Hemoglobin, which should appear firm, translucent, and light tan in color.

BIBLIOGRAPHY: 1. Faru, Y. C., et al., Health Lab. Scien., 10:55, 1973.

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TECHNICAL DATA SHEET #230 REV. 4 2. 3. 4. 5. 6. 7. 8. 9.

Forbes, B. A., et al., Bailey and Scott’s Diagnostic Microbiology, 12th ed., C. V. Mosby, St. Louis, 2007. Hipp, S. S., et al., Appl. Microbiol., 27:192, 1974. Hipp, S. S., et al., J. Clin. Pathol., 1:476, 1975. Johnson, J. Venereal Dis. Inform., 26:239, 1945. Martin, J. E., et al., Appl. Microbiol., 27:802, 1974. Martin, J. E., and A. Lester, HSMHA Health Reports, 86:30, 1971. Martin, J. E., and J. E. Lewis, Publ. Health Lab., 35:33, 1977. Murray, P.R., et al., Manual of Clinical Microbiology, 9th ed., American Society for Microbiology, Washington, D.C., 2007. 10. Odegard, K., Acta Path. Scand., Section B, 79:545-548, 1971. 11. Personal Communication, USHEW, CDC, Atlanta, 1979. 12. Riddell, R. H., and A. C. Buck, J. Clin. Pathol., 23:481-483, 1970. 13. Robinson, R. Q., Memorandum, USHEW, CDC, January 2, 1975. 14. Seth, A., Br. J. Ven. Dis., 46:201-202, 1970. 15. Thayer, J. D., and J. E. Martin, Publ. Health Reports, 79:49, 1964. 16. Thayer, J. D., and J. E. Martin, Publ. Health Reports, 81:559, 1966. *For more detailed information consult appropriate references. bioMérieux, Inc. Data #230 Revision Date: May 2009

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