Chocolate Agar: Composition, uses and colony characteristics

Haemophilus influenzae on chocolate agar

Chocolate Agar (CAP) is the lysed blood agar. The name is itself derived from the fact that red blood cell (RBC) lysis gives the medium a chocolate-brown color. It is used  for the isolation of fastidious organisms, such as Haemophilus influenzae, when incubated at 35-37°C in a 5%
CO2 atmosphere.

The composition of chocolate agar and the Blood Agar is same and the only difference is while preparing Chocolate agar, the red blood cells are lysed.

Haemophilus influenzae on chocolate agar
Haemophilus influenzae on chocolate agar

The lysis of RBC during the heating process releases intracellular coenzyme Nicotinamide adenine dinucleotide (NAD or V Factor) into the agar for utilization by fastidious bacteria (the heating process also inactivates growth inhibitors). Hemin (factor X)  is available from non-hemolyzed as well as hemolyzed blood cells.

The most common species that require this enriched medium for growth include Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus spp. Neither of these species is able to grow on sheep blood agar.

Preparation of Chocolate Agar:

  1. Heat-lyse a volume of horse or sheep blood that is 5% of the total volume of media being prepared very slowly to 56°C in a water bath.
  2. Dispense 20 ml into 15×100 mm Petri dishes. Allow the media to solidify and condensation to dry.
  3. Place the plates in sterile plastic bags and store at 4ºC until use.
  4. As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with ~5% CO2 (or in a candle-jar).

Quality Control:

  1. Grow N. meningitidis, S. pneumoniae, and H. influenzae QC strains for 18-24 hours on a CAP at 35-37°C with ~5% CO2 (or in a candle-jar).
  2. Observe the CAP for specific colony morphology and hemolysis.

Passing result:

  • N. meningitidis and H. influenzae should appear as large, round, smooth, convex, colorless-to grey, opaque colonies on the CAP with no discoloration of the medium.
  • S. pneumoniae should appear as small grey to green colonies with a zone of alpha-hemolysis (only slightly green) on the CAP.
  • After 48 hours, the sterility test plate should remain clear

Colony characteristics in chocolate agar

  1. Neisseria meningitidis: Growth on chocolate agar is grayish, non-hemolytic, round, convex, smooth, moist, glistening colonies with a clearly defined edge.
  2. Neisseria gonorrhoeae: Colonies on Chocolate agar are pinkish-brown and translucent, exhibit smooth consistency and defined margins, and are typically 0.5-1 mm in diameter.
  3. Haemophilus influenzae: Non hemolytic, opaque cream-to-gray colonies (accompanying Sheep blood agar shows no growth)

Modification of Chocolate Agar: 

  1. Thayer-Martin agar: It is used for the selective isolation of N. gonorrhoeae and N. meningitidis. Thayer-Martin Media is a chocolate agar supplemented with vancomycin, nystatin and colistin to inhibit the normal flora, including nonpathogenic Neisseria.
  2. Chocolate Agar with bacitracin: CAP with bacitracin is a selective medium used to improve the primary isolation of H. influenzae from specimens containing a mixed flora of bacteria and/or fungi.
  3. Chocolate agar with GC base and growth supplement: It is a medium that supports the special growth requirements (hemin and NAD) needed for the isolation of fastidious organisms, such as H. influenzae, when incubated at 35-37°C in a 5% CO2 atmosphere
  4. Chocolate agar with TSA and growth supplements: It is a medium that supports the special growth requirements (hemin and NAD) needed for the isolation of fastidious organisms, such as H. influenzae, when incubated at 35-37°C in a 5% CO2 atmosphere.
About tankeshwar 386 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion, I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. My name is Callistus from federal university Bayelsa state, Nigeria. Am a final year microbiology student. Do u have provisions for a masters degree program in epidemiology and biostatistics in your institution? And are there scholarship opportunities?

  2. May name is Moise NTIGURIRWA from INES-Ruhengeri (RWANDA) and I need more about N. gonorrhea and Chocolate Agar.

Do you have any queries? Please leave me in the comments section below. I will be happy to read your comments and reply.

This site uses Akismet to reduce spam. Learn how your comment data is processed.