Cat. no. E11 Chocolate with Bacitracin, 15x100mm Plate, 18ml 10 plates/bag


Hardy Diagnostics Chocolate Agar with Bacitracin is recommended for use in the isolation and cultivation of Haemophilus species from respiratory specimens containing mixed flora.


Chocolate Agar with Bacitracin consists of GC Agar Base with added hemoglobin and KoEnzyme Enrichment. GC Agar Base contains proteose peptone which provides nitrogenous nutrients. When heated, hemoglobin releases hemin (X-factor), a factor required by fastidious organisms such as Haemophilus species. The phosphate buffer is added to maintain the pH of the medium. Corn starch aids in neutralizing any toxic fatty acids present in the medium. KoEnzyme Enrichment is a chemically defined enrichment that provides NAD (V-factor), amino acids, vitamins, dextrose, ferric ions, and coenzymes which promote growth. Bacitracin inhibits most strains of streptococci, staphylococci, Neisseria and Micrococcus species. Because Haemophilus spp. are resistant to the concentration of bacitracin employed in this medium, Chocolate Agar with Bacitracin allows for their increased recovery, due to the decreased competition for nutrients in the medium.


Ingredients per liter of deionized water:*

Chocolate Agar with Bacitracin:
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
Bacitracin 20,000U
Agar 10.0gm
KoEnzyme Enrichments:
Dextrose 10.0gm
L-Cystine 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 B 12 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), 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 medium 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. Examine plate for typical colonial morphology and characteristics.


Consult listed references for the interpretation of growth of fastidious species. (1-3,5,7)


E. coli , some Neisseria species, strains of Candida spp., Klebsiella , Proteus , and Pseudomonas spp., as well as other bacteria may grow on this medium.


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
Haemophilus influenzae
ATCC ® 10211
A 24hr 35°C CO 2 ** Growth
Staphylococcus epidermidis
ATCC ® 12228
B 24hr 35°C Aerobic Inhibited

User Quality Control

** Atmosphere of incubation is enriched with 5-10% CO 2 .


Chocolate Agar with Bacitracin should appear opaque, and brown in color.

H. influenzae growing on Chocolate Agar with Bacitracin

Haemophilus influenzae (ATCC ® 10211) colonies growing on Chocolate Agar with Bacitracin (Cat. no. E11). Incubated in CO 2 for 24 hours at 35ºC.

S. epidermidis inhibited on Chocolate Agar with Bacitracin

Staphylococcus epidermidis (ATCC ® 12228) growth inhibited on Chocolate Agar with Bacitracin (Cat. no. E11). Incubated aerobically for 24 hours at 35ºC.


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. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., 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. Maritn, 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-A2, Vol. 16, No.16. 1996. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

ATCC is a registered trademark of the American Type Culture Collection.