Chocolate Agar Composition Uses

6 September, 2025

Introduction

Chocolate Agar (CA) is an enriched culture medium derived from blood agar. Specifically, it is prepared by heating blood agar until the red blood cells lyse, releasing intracellular nutrients such as hemoglobin (factor X) and NAD (factor V).

As a result, the medium gets its brown “chocolate-like” color due to the lysis of red blood cells. Moreover, it is particularly important for the growth of fastidious bacteria like Haemophilus influenzae and Neisseria gonorrhoeae, which do not grow well on plain blood agar

Media Type

Category: Enriched medium
Differential? No (unlike blood agar, hemolysis cannot be detected because RBCs are already lysed)
Purpose: For growth of fastidious organisms requiring X and V factors.

Composition & pH of Chocolate Agar

Chocolate agar is essentially nutrient agar + blood (heated to lyse RBCs).

Component	Function
Peptone (5 g)	Source of amino acids & nitrogen
Beef extract (3 g)	Provides vitamins & salts
Sodium chloride (5 g)	Maintains osmotic balance
Agar (15 g)	Solidifying agent
Distilled water (1 L)	Solvent
Defibrinated blood (50–100 mL)	Source of growth factors X & V

Final pH: 7.2 – 7.4 (at 25°C)

Sterilization

Nutrient agar base: Autoclaved at 121°C for 15 minutes.
Blood: Added aseptically and heated to lyse RBCs.

Preparation of Chocolate Agar

Prepare nutrient agar base and sterilize.
Cool to 45–50°C.
Add 5–10% sterile defibrinated blood.
Heat the mixture to 75–80°C until the medium turns brown (chocolate color) due to RBC lysis.
Mix gently and pour into sterile Petri dishes.
Allow to solidify and store at 2–8°C.

Uses of Chocolate Agar

Growth of fastidious organisms requiring X (hemin) and V (NAD) factors.
- Haemophilus influenzae (requires both X & V factors).
- Neisseria gonorrhoeae and Neisseria meningitidis (require V factor).
Commonly used in:
- Thayer-Martin medium → Selective chocolate agar with antibiotics (for Neisseria gonorrhoeae).
- Modified chocolate agar for isolation of Haemophilus species.
For clinical specimens:
- Respiratory infections (e.g., Haemophilus influenzae from sputum).
- Genital specimens (e.g., Neisseria gonorrhoeae).
- CSF samples (e.g., Neisseria meningitidis in meningitis).

Limitations

More expensive compared to nutrient or blood agar.
Cannot differentiate hemolysis (since RBCs are lysed).
Requires careful aseptic preparation.
Some strains may require enrichment with antibiotics or additional growth factors.

Conclusion

Chocolate agar is a vital enriched medium in diagnostic microbiology. By providing X and V factors, it supports the growth of important fastidious pathogens like Haemophilus influenzae and Neisseria gonorrhoeae.

Category: Uncategorized