Chocolate agar supports the growth of fastidious bacteria that can’t survive on standard blood agar, most notably Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis. It also grows many of the same organisms that regular blood agar supports, making it a broadly useful medium in clinical microbiology labs. The name comes from its brown, chocolate-like color, not from any cocoa content.
Why Chocolate Agar Exists
Some bacteria need specific nutrients that are locked inside red blood cells. On standard blood agar, those cells remain intact, so the nutrients stay out of reach. Chocolate agar solves this by heating defibrinated blood (usually from sheep, horses, or goats) to above 60°C for 15 to 20 minutes. That heat bursts the red blood cells open, releasing two critical growth factors: hemin (called Factor X) and a coenzyme called NAD (Factor V). These two compounds are essential for growing certain bacteria that simply won’t appear on unheated blood agar.
Haemophilus influenzae
Haemophilus influenzae is the classic reason chocolate agar exists. This bacterium has an absolute requirement for both hemin and NAD during aerobic growth, and it cannot make either on its own. On regular blood agar, intact red blood cells hold these factors hostage. On chocolate agar, both are freely available in the medium, allowing Haemophilus to thrive. This makes chocolate agar the go-to medium for isolating Haemophilus from respiratory specimens, cerebrospinal fluid, and other clinical samples.
Neisseria Species
The two most clinically important Neisseria species, N. gonorrhoeae (the cause of gonorrhea) and N. meningitidis (a cause of bacterial meningitis), both grow well on chocolate agar. These organisms are picky about their environment. They require incubation at 37°C in a 5% CO₂ atmosphere. Without that supplemental carbon dioxide, Neisseria species show no growth on standard chocolate agar at all. With it, heavy growth appears within 24 hours.
Colony appearance can help identify which Neisseria species is present. Non-piliated colonies tend to be flat, round, and smooth-edged. Piliated colonies (those with hair-like surface structures that help bacteria attach to human tissue) are smaller, domed, and have irregular or pointed edges with a visible ring at the border.
When labs need to isolate Neisseria from sites with lots of other bacteria, like the throat, rectum, or urethra, they use a selective version of chocolate agar called Thayer-Martin agar. This adds antibiotics (vancomycin, colistin, nystatin, and trimethoprim) to suppress normal flora and saprophytic Neisseria while letting the pathogenic species grow through.
Moraxella catarrhalis
Moraxella catarrhalis, a common cause of ear infections, sinusitis, and respiratory infections in people with chronic lung disease, grows readily on chocolate agar. Its colonies have a distinctive physical property: they can be pushed across the agar surface intact, sliding like a hockey puck on ice without breaking apart. This “hockey puck sign” is a quick way to distinguish Moraxella from Neisseria colonies, which look similar but don’t slide the same way.
Other Organisms That Grow
Chocolate agar is not limited to fastidious bacteria. Because it contains all the nutrients of regular blood agar plus the released intracellular factors, many common organisms grow on it too. Streptococcus pneumoniae, various Staphylococcus species, and Escherichia coli will all produce colonies. The difference is that these organisms don’t need chocolate agar; they grow just fine on simpler media. Chocolate agar is chosen specifically when a lab suspects organisms that require those extra growth factors, or when processing sterile-site specimens like spinal fluid or joint fluid where missing a fastidious pathogen would be a serious problem.
What Won’t Grow
Chocolate agar is a non-selective, enriched medium, so it’s permissive to many species. However, it won’t support obligate anaerobes (bacteria that die in the presence of oxygen), since it’s incubated aerobically. It also won’t reliably grow organisms with extremely specific requirements beyond what heated blood provides, such as Legionella (which needs charcoal-based media) or Mycobacterium tuberculosis (which needs specialized media like Löwenstein-Jensen).
How Chocolate Agar Is Prepared
The base medium is typically a gonococcal agar or GC base dissolved in water and autoclaved at 121°C. After sterilization, the base is cooled to around 80 to 87°C, and defibrinated blood is added aseptically. Gentle shaking for 15 to 20 minutes lyses the red blood cells, turning the medium from red to a uniform dark brown. The temperature has to be high enough to rupture the cells and release hemin and NAD, but the medium is cooled to around 55°C before pouring into plates so the heat doesn’t destroy more delicate supplements that may be added, like bacitracin or other growth enhancers.
In resource-limited settings, human blood is sometimes used, though this produces poorer bacterial isolation. Antibodies naturally present in human blood can actually inhibit the growth of the very organisms the medium is designed to support. Sheep and horse blood remain the preferred sources worldwide.
Incubation Conditions That Matter
Plating bacteria on chocolate agar is only half the equation. The incubation environment determines whether fastidious organisms actually grow. For Neisseria species, a 5% CO₂ incubator at 37°C is essential. Without supplemental CO₂, these bacteria produce zero colonies on standard chocolate agar. Research has shown that adding sodium bicarbonate directly to the agar can substitute for a CO₂ incubator, producing moderate growth at 24 hours and heavy growth by 48 hours in normal atmospheric conditions. This is a practical consideration for labs in settings where CO₂ incubators aren’t available.
Haemophilus influenzae also benefits from CO₂ enrichment, though it’s somewhat less dependent on it than Neisseria. Most clinical labs incubate chocolate agar plates at 35 to 37°C in 5% CO₂ as a default, covering the needs of all the fastidious organisms the medium is designed to support.