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. H25 Chocolate Agar, 15x150mm Plate, 69ml 10 plates/bag
Cat. no. J42 Blood Agar, 5% / Chocolate Agar, 15x100mm Biplate,
10 plates/bag
Cat. no. J44 Chocolate Agar / Martin Lewis Agar with Lincomycin,
15x100mm Biplate, 10ml/10ml
10 plates/bag
Cat. no. J72 Chocolate Agar / Modified Thayer Martin (MTM) Agar,
15x100mm Biplate, 10ml/10ml
10 plates/bag
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:*

Chocolate Agar:
Proteose Peptone 15.0gm
Sodium Chloride 5.0gm
Dipotassium Phosphate 4.0gm
Monopotassium Phosphate 1.0gm
Corn Starch 1.0gm
Hemoglobin, Bovine 10.0gm
KoEnzyme Enrichment 10.0ml
Agar 10.0gm
KoEnzyme Enrichment:
Dextrose 10.0gm
L-Cysteine, HCl 2.59gm
L-Glutamine 1.01gm
L-Cystine 0.11gm
Adenine 0.101gm
Nicotinic Adenine Dinucleotide 25.0mg
Cocarboxylase 10.0mg
Guanine Hydrochloride 3.0mg
Ferric Nitrate 2.0mg
P-Aminobenzoic Acid 1.3mg
Vitamin B12 1.0mg
Thiamine 0.3mg

Final pH 7.2 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


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% CO2 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 CO2 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.


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.

Precipitated hemoglobin may appear as dark spots on or in the media and does not affect the performance of the media.

The presence or absence of N. gonorrhoeae in a specimen does not rule out the possible presence of other pathogenic organisms.


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
Time Temperature Atmosphere
Neisseria gonorrhoeae
ATCC® 43069
A 24hr 35°C CO2** Growth
Haemophilus influenzae
ATCC® 10211
A 24hr 35°C CO2** Growth

** Atmosphere of incubation is enriched with 5-10% CO2.

User Quality Control


Chocolate Agar should appear opaque, and brown in color.

H. influenzae growing on Chocolate Agar

Haemophilus influenzae (ATCC® 10211) colonies growing on Chocolate Agar (Cat. no. E14) Incubated in CO2 for 24 hours at 35ºC.

N. gonorrhoeae colonies growing on Chocolate Agar

Neisseria gonorrhoeae (ATCC® 43069) colonies growing on Chocolate Agar (Cat. no. E14) Incubated in CO2 for 24 hours at 35ºC.

Chocolate Agar

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.