Chocolate agar foundation, uses and preparation

Simon Doyle

The chocolate agar It is a solid, enriched, non-selective and non-differential culture medium. It is used mainly for the isolation of demanding microorganisms from the nutritional point of view, although any type of bacteria can grow in it..

That is why its usefulness increases especially in the seeding of samples that are normally sterile, such as CSF and joint fluid. Although it is also included within the chosen means for sowing polymicrobial samples, but in these cases it is necessary to add antibiotics that inhibit the concomitant flora.

Chocolate agar with bacterial growth

This medium has a characteristic brown color very similar to chocolate, hence its name. The preparation is very similar to that of blood agar, only in this case the blood must be heated in order for the red blood cells to break down..

Its preparation, like blood agar, is very delicate, as it is easily contaminated. For this reason, many laboratories prefer to acquire this medium already prepared by commercial companies that guarantee its quality..

Article index

  • 1 Rationale
  • 2 Uses
    • 2.1 Chocolate agar prepared with Columbia agar
    • 2.2 Chocolate agar prepared with GC base agar (for gonococci)
    • 2.3 Chocolate agar prepared with Müeller Hinton agar
    • 2.4 Chocolate agar prepared with Thayer Martin agar
  • 3 Preparation
    • 3.1 Calculations
    • 3.2 Weigh and dissolve
    • 3.3 Sterilize
    • 3.4 Blood aggregation
    • 3.5 Another way to prepare chocolate agar without using blood
  • 4 Quality control
  • 5 References


This medium consists of a nutrient-rich agar base and heated blood. Hemolysis of red blood cells provide factor X (hemin) and factor V (NAD) to the medium, necessary for the growth of some microorganisms, such as the genus Haemophilus. It is also very useful for isolating Neisserias sp.

Like blood agar, different media can be used as base agar depending on the need. Among the media used are brain heart infusion and trypticase soy agar, although the most recommended are Columbia agar, Müeller Hinton, GC agar and Thayer Martin agar..

Some variants of chocolate agar include a commercially available fortified supplement called Isovitalex or Polivitex..

These supplements contain vitamin B12, L-glutamine, adenine, guanine hydrochloride, p-aminobenzoic acid, nicotinamide adenine dinucleotide (NAD), thiamine pyrophosphate, ferric nitrate, thiamine hydrochloride, cysteine ​​hydrochloride, L-cystine and glucose.

It is important to note that chocolate agar is more enriched than blood agar, but it does not allow the observation of hemolysis patterns..


Source: Author:
Pete Seidel, Amanda Moore, MT, Todd Parker, PhD, Audra Marsh, USCDCP

Chocolate agar prepared with Columbia agar

This medium contains pancreatic digest of casein and heart, peptic digest of meat, sodium chloride, agar, yeast extract and cornstarch. It is also rich in vitamins, minerals and essential amino acids.

This heated blood base is ideal for the isolation of bacteria of the genus Neisseria. On the other hand, if a supplement for Brucella is additionally added to the medium, the aforementioned microorganism can be isolated. Results are improved using horse blood.

Chocolate agar prepared with GC base agar (for gonococci)

This medium contains peptones, cornstarch, monobasic and dibasic buffers, sodium chloride, and agar..

Most commercially prepared chocolate agar presentations come with this base and do not contain heated blood, but instead a synthetic mixture of hemin and a chemical supplement of growth factors, vitamins, minerals, amino acids, factor V and glucose..

Chocolate agar prepared with Müeller Hinton agar

It is used to perform antimicrobial susceptibility testing of fastidious microorganisms, such as Streptococcus pneumoniae using 5% heated ram blood.

It is also used for the primary isolation of Neisserias and Haemophilus, but in the particular case of the isolation of Haemophilus The use of horse blood is preferred, as it is a rich source of factor X and V.

On the other hand, if the sample to be sown comes from a non-sterile area, the addition of antibiotics is recommended to inhibit the normal flora of the area..

Example for respiratory samples where the presence of bacteria of the genus is suspected Haemophilus Bacitracin is used to inhibit the growth of Staphylococcus, Micrococcus, Streptococcus and saprophytic Neisserias.

In the case of genital chancre samples, where it is suspected of Haemophilus ducreyi, A chocolate agar prepared as follows has been successfully used: Müeller - Hinton agar with 5% chocolateized horse blood, 1% Isovitalex enrichment and 3 µg / ml vancomycin.

Chocolate agar prepared with Thayer Martin agar

This medium is special for the isolation of Neisseria gonorrhoeae. It must contain antibiotics to inhibit the accompanying flora. Lamb's blood is used.


You should see the indications for the preparation of the base agar to be used. They are found on the back of the container of the dehydrated medium. They generally describe how much you must weigh to prepare a liter of culture medium.

In the laboratory, the exact amount needed can be prepared, it can be more or less than a liter.


A rule of three is used to calculate how much to weigh to prepare the desired volume. Example:

If for 1 liter it is necessary to weigh 40 g and the laboratory requires 800 ml, it is said:

1000 ml -40 gr

800 ml - X

The formula will be as follows:

X = 32 gr (quantity to be weighed for 800 ml).

Weigh and dissolve

The necessary quantity is weighed and placed in a flask with the water.

Heat over moderate heat and mix gently with rotary movements until the dehydrated medium completely dissolves, allowing it to boil for 1 minute..


The flask is placed in the autoclave to sterilize the medium at 121 ° C for 20 minutes..

Blood aggregate

When leaving the autoclave, let it rest until the temperature of the medium is approximately between 56 to 70 ° C to place the blood and mix until the medium turns brown..

If you are adding supplements, this is the time to do it. Subsequently mix and serve 20 ml to each sterile Petri dish.

The entire procedure should be done in a laminar flow hood or around the Bunsen burner..

Let stand until solid and store inverted in the fridge.

Another way to prepare chocolate agar without using blood

The base medium is prepared as described above, the commercially obtained dehydrated hemoglobin is dissolved and sterilized in the autoclave..

Both solutions are allowed to cool to 50 ° C, are joined and the supplement is added. Mix under aseptic conditions and then serve in sterile Petri dishes..


It is important that the blood is placed at the indicated temperature, as it is the ideal one to lyse the red blood cells and at the same time maintain factor V, which is temperature sensitive..

No bubbles should remain on the surface of the agar. From each batch of 100 plates, one or two plates should be incubated in the oven at 37 ° C for 24 hours to check their sterility..

For best results, chocolate agar should be used shortly after preparation..

Control bacterial strains should be kept in the laboratory to evaluate the efficacy of the fresh medium for the growth of the main fastidious microorganisms of clinical importance..


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  4. Llanes R, Reyes A, Rodríguez C, Guzmán D, Llop A. Feasibility of using the GC-Biocen Agar base culture medium in the Microbiology Laboratory. Rev Cubana Med Trop,  2004; 56 (3): 237-238. Available at: scielo.sld.
  5. Wikipedia contributors. Chocolate agar. Wikipedia, The Free Encyclopedia. December 17, 2018, 19:54 UTC. Available at:

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