Chocolate Agar: Composition, Preparation, Uses

Haemophilus influenzae on chocolate agar
Haemophilus influenzae on chocolate agar

Chocolate Agar (CAP or CHOC) is a nonselective, enriched medium used for the isolation and identification of fastidious pathogens. Chocolate agar is prepared by heating blood agar, which in turn ruptures the red blood cell (RBC) and releases nutrients that aid in the growth of fastidious bacteria, most notably Haemophilus and Neisseria species. The name is derived from the fact that the lysis of RBC gives the medium a chocolate-brown color.


In-house prepared chocolate agar is modified blood agar which is heated to lyze the RBC. The lysis of RBC releases intracellular nutrients such as hemoglobin, hemin (X factor), and the coenzyme nicotinamide adenine dinucleotide (NAD or V factor) into the agar for utilization by fastidious bacteria. Peptone provides the organism with nitrogen, amino acids, and other elements essential for growth, sodium chloride maintains the osmotic balance, and agar acts as a solidifying agent.

In commercial preparation, casein and animal tissue digest provides the organism with nitrogen, amino acids, and other elements. Since Neisseria species are highly sensitive to toxic substances such as fatty acids; cornstarch is added to neutralize possible toxic metabolites. Potassium phosphate helps to maintain a uniform pH during growth. Hemoglobin solution provides X factor (hemin) required by Haemophilus species, and isovitox enrichment provides V-factor (nicotinamide dinucleotide), and other complex compounds that enhance the growth of Neisseria species.

Composition of Chocolate Agar

The composition of chocolate agar is the same as that of blood agar. The only difference is blood agar is heated in a water bath to prepare chocolate agar. Commercially available chocolate agar medium, however, has a different composition and requires no addition of blood

Casein/animal tissue digest15.0
Potassium phosphate, dibasic4.0
Potassium phosphate, monobasic1.0
Sodium chloride5.0
Hemoglobin solution (2%)500.0 ml
Isovitox enrichment10.0 ml

Preparation of Chocolate Agar

  1. Prepare the blood agar base as instructed by the manufacturer.
  2. Sterilize by autoclaving at 121°C for 15 minutes.
  3. Add 5-7% v/v of defibrinated blood (horse or sheep blood) and place the media in a water bath of 75 -80°C, and keep swirling gently until the color changes to dark brown.
  4. Pour into sterile Petri plates under aseptic conditions after the media has cooled to 50-55°C.
  5. Label the plates with the name of the media, and date of preparation, and store them invertedly at 2-8°C until use.
Chocolate agar slants

Note: Other supplements such as isovitalex could be added during preparation.

Chocolate agar slants

  1. Dispense 4 mL of the medium into 16X125 mm screw-cap tubes
  2. Keep the tubes in a slanted position and let them solidify.

Chocolate agar slants appear brown to brownish-red color. They should be stored at 4°C when not in use and warmed to room temperature (25°C) before use.

Colony Morphology

Almost all organisms grow on chocolate agar giving grey colonies of various sizes. However, since it is used specifically to isolate fastidious pathogens such as Neisseria and Haemophilus, their colony morphologies are described below.

OrganismColony morphology
Neisseria gonorrhoeaepinkish-brown and translucent, exhibit smooth consistency and defined margins, and are typically 0.5-1 mm in diameter
N. meningitidisgrayish, non-hemolytic, round, convex, smooth, moist, glistening colonies with a clearly defined edge, larger in size as compared to N. gonorrhoeae
Haemophilus influenzaeNonhemolytic, colorless, moist colonies with a characteristic “mousy” odor.

Quality Control

Perform sterility testing of the prepared media by incubating 3-5 uninoculated plates of each batch at 37°C for 18-24 hours. Any growth should be regarded as positive, and the whole lot should be discarded.

Performance testing of the media should be done by inoculating the following organisms to prepared plates and incubating them for 18-24 hours at 35-37°C with 5% CO2 (or in a candle jar but it can only provide up to 3% CO2) to determine the growth performance of the completed medium

OrganismExpected Result
Neisseria gonorrhoeae ATCC 43069Luxuriant growth, small, grey to white colonies
Haemophilus influenzae ATCC 10211Good  growth, small, colorless, moist colonies

Uses of Chocolate Agar

  1. To isolate fastidious organisms such as H. influenzae, N. gonorrhoeae, and N. menigitidis from various clinical specimens, that aid in the diagnosis of disease.
  2. Chocolate agar with bacitracin acts as a selective medium for screening H. influenzae from specimens e.g. sputum containing a mixed flora of microorganisms

Limitations of Chocolate Agar

  1. Since chocolate agar is an enriched medium, it gets contaminated very easily.
  2. Use of chocolate agar only, makes it difficult to differentiate non-pathogenic organisms from pathogenic ones as the former overgrow pathogenic bacteria.
  3. Chocolate agar has a reduced concentration of agar, so the surface of the plate is fragile and prone to scratches during streaking.

Modifications of Chocolate Agar

  1. Thayer-Martin Media: Thayer martin medium is a modification of chocolate agar supplemented with vancomycin, nystatin, and colistin to inhibit the normal flora, including nonpathogenic Neisseria for the selective isolation of N. gonorrhoeae and N. meningitidis. 
  2. Chocolate Agar with bacitracin: This modification is used as a selective medium to improve the primary isolation of H. influenzae from specimens such as sputum that contains a mixed flora of bacteria and/or fungi.
  3. Chocolate agar supplemented with GC base and growth supplement: This variant of chocolate agar is used to support the special growth requirements (hemin and NAD) needed for the isolation of Haemophilus spp. when incubated at 35-37°C in a 5% CO2 atmosphere.
  4. Chocolate agar with TSA and growth supplements: It is a modification of chocolate agar 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.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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