Chocolate Agar: Composition, uses and colony characteristics

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

The lysis of RBC during the heating process  releases intracellular coenzyme Nicotinamide adenine dinucleotide (NAD or V Factor) in to 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:

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.