|Cat. no. D21||Chocolate Agar, 15x45mm Tube, 1.8ml Slant||100 tubes/box|
|Cat. no. E14||Chocolate Agar, 15x100mm Plate, 19ml||10 plates/bag|
|Cat. no. E14BX||Chocolate Agar, 15x100mm Plate, 19ml||100 plates/box|
|Cat. no. E452||Chocolate Agar, 50x75mm Square Pill Pocket Plate, 16ml||10 plates/bag|
|Cat. no. H25||Chocolate Agar, 15x150mm Plate, 69ml||10 plates/bag|
|Cat. no. J42||
Blood Agar, 5% / Chocolate Agar, 15x100mm Biplate,
|Cat. no. J44||
Chocolate Agar / Martin Lewis Agar with Lincomycin,
15x100mm Biplate, 10ml/10ml
|Cat. no. J72||
Chocolate Agar / Modified Thayer Martin (MTM) Agar,
15x100mm Biplate, 10ml/10ml
|Cat. no. L37||Chocolate Agar, 16x100mm Tube, 5.5ml Slant||20 tubes/box|
Hardy Diagnostics Chocolate Agar is recommended for use in the isolation and cultivation of fastidious microorganisms, particularly Haemophilus and Neisseria species.
In the late 1920s, McLeod et al. developed Chocolate Agar using a formulation incorporating yeast extract and peptones. (9) The development of this medium provided the ground work for the improvement of culture methods for fastidious microorganisms. In 1945, Johnston reported a medium which produced Neisseria gonorrhoeae colonies successfully within 24 hours. (6) The recovery time of N. gonorrhoeae was further decreased by Carpenter and Morton in 1947 when they added GC Agar Base, enriched with hemoglobin and yeast extract, to the Chocolate Agar formulation. (4) The accelerated growth of N. gonorrhoeae was attributed to differences in gel strength, thus the new formulation contained less agar than previously indicated. (6) The medium was further improved in 1967 by Martin et al. by replacing the yeast concentrate with a chemically defined enrichment (KoEnzyme Enrichment) designed to aid the growth of Neisseria species. (8)
Chocolate Agar consists of GC Agar Base with added hemoglobin and KoEnzyme Enrichment. GC Agar Base contains proteose peptone which provides nitrogenous nutrients. Hemoglobin releases hemin (X-factor) components. The phosphate buffer is added to maintain the pH of the medium. Corn starch aids in neutralizing any toxic fatty acids present. KoEnzyme Enrichment is a chemically defined supplement that provides NAD (V-factor), amino acids, vitamins, dextrose, ferric ions, and coenzymes to promote the growth of Neisseria species.
Ingredients per liter of deionized water:*
|Nicotinic Adenine Dinucleotide||25.0mg|
|Vitamin B 12||1.0mg|
Final pH 7.2 +/- 0.2 at 25ºC.
* Adjusted and/or supplemented as required to meet performance criteria.
STORAGE AND SHELF LIFE
Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.
Specimen Collection: Consult listed references for information on specimen collection. (1-3,5,7) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and maintained at an appropriate temperature. (1-3,5,7)
Method of Use: Inoculate the Chocolate Agar and streak the specimen as soon as possible after it is received in the clinical laboratory. If the specimen is being cultured directly from a swab, roll the swab over a small portion of the agar surface, and streak for isolation. Incubate in 5-10% CO 2 at 35-37ºC. for 24 to 48 hours. Extend incubation if needed. Subcultures of N. gonorrhoeae should be made within 18-24 hours.
Square Pill Pocket Plate: After inoculation, place one CO 2 tablet in the pill pocket and place plate in a sealed zip bag. Do not invert the plate. Proceed with incubation parameters as outlined above.
INTERPRETATION OF RESULTS
Consult listed references for the interpretation of growth of fastidious species. (1-3,5,7)
Chocolate Agar is an enriched medium, thus non-pathogenic organisms may overgrow pathogenic bacteria. If isolation of N. gonorrhoeae is desired, a selective medium such as Thayer Martin Agar, Modified (Cat. no. E30), or Martin Lewis Agar with Lincomycin (Cat. no. E39) should be used in parallel with this non-selective formula.
The presence or absence of N. gonorrhoeae in a specimen does not rule out the possible presence of other pathogenic organisms.
MATERIALS REQUIRED BUT NOT PROVIDED
Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.
|Test Organisms||Inoculation Method*||Incubation||Results|
ATCC ® 43069
|A||24hr||35°C||CO 2 **||Growth|
ATCC ® 10211
|A||24hr||35°C||CO 2 **||Growth|
** Atmosphere of incubation is enriched with 5-10% CO 2 .
User Quality Control
Chocolate Agar should appear opaque, and brown in color.
Haemophilus influenzae (ATCC ® 10211) colonies growing on Chocolate Agar (Cat. no. E14) Incubated in CO 2 for 24 hours at 35ºC.
Neisseria gonorrhoeae (ATCC ® 43069) colonies growing on Chocolate Agar (Cat. no. E14) Incubated in CO 2 for 24 hours at 35ºC.
Uninoculated plate of Chocolate Agar (Cat. no. E14).
1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.
2. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.
3. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.
4. Carpenter, C.M. and H.E. Morton. 1947. Proc. N.Y. State Assoc. Public Health Labs ; 27:58-60.
5. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
6. Johnston, et al. 1945. J. Ven. Dis. Inf. ; 26:239.
7. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.
8. Martin, J.E., et al. 1967. Public Health Rep. ; 82:361.
9. McLeod, J.W., et al. 1927. Br. J. Exp. Pathol. ; 8:25.
10. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.
ATCC is a registered trademark of the American Type Culture Collection.