Bile Esculin Agar
Presumptive identification of enterococci and group D streptococci
Bile Esculin Agar is a selective and differential medium that identifies enterococci and group D streptococci by two properties: growth in the presence of bile and hydrolysis of esculin. Positive organisms turn the medium around their colonies dark brown to black. The formulation follows the bile esculin test described by Swan (1954) and Facklam and Moody (1970).
Also known as: BEA, Bile Aesculin Agar, bile esculin medium.
Principle of the Medium
Oxgall inhibits most Gram-positive bacteria, but enterococci and group D streptococci tolerate bile and grow. Organisms that hydrolyse esculin release esculetin, which reacts with ferric citrate to form a dark brown to black complex. Many Gram-negative bacteria can also grow, but most do not blacken the medium.
Oxgall suppresses most other Gram-positive organisms
Esculetin and ferric ions form a brown-black complex
Both properties together give the presumptive result
Typical Reactions
| Organism | Growth | Blackening |
|---|---|---|
| Enterococcus faecalis, E. faecium | Good | Positive |
| Streptococcus gallolyticus (S. bovis group) | Good | Positive |
| Streptococcus pyogenes and other non-group D streptococci | Inhibited or poor | Negative |
| Proteus mirabilis and many Gram-negative rods | Good | Negative |
| Some viridans streptococci, Listeria, Aerococcus, Leuconostoc, Pediococcus | Variable | May be positive |
Comparable Products
| Supplier | Product | Relationship |
|---|---|---|
| AuSaMicS | Bile Esculin Agar (AS-1140), 43.5 g/L | — |
| BD Difco | Bile Esculin Agar | Same published formulation |
| HiMedia | Bile Esculin Agar (M972), 64.5 g/L | Comparable published formulation; higher bile content |
Based on published formulations. Trademarks belong to their respective owners.
Advantages and Limitations
Advantages
- ✓ Clear visual result within 24–48 h
- ✓ Well-established test in use since the 1950s
- ✓ Azide-free, so it can be used for slants and plates
- ✓ Simple preparation: one powder, standard autoclaving
Limitations
- ▲ Presumptive only: confirm with further tests
- ▲ Not selective against Gram-negatives
- ▲ Other esculin-positive, bile-tolerant organisms can react
- ▲ Does not separate enterococci from S. gallolyticus
Frequently Asked Questions
What is Bile Esculin Agar used for?
It is used to presumptively identify enterococci and group D streptococci. These organisms grow in the presence of bile and hydrolyse esculin, which turns the medium dark brown to black.
What does a positive bile esculin test look like?
On plates, a dark brown to black zone forms around the colonies. On slants, more than half of the slant turns black within 48 hours.
Does Bile Esculin Agar contain sodium azide?
No. AS-1140 is azide-free. Bile Esculin Azide Agar, used in ISO 7899-2 for water testing, is a different medium that contains sodium azide.
Should Bile Esculin Agar be autoclaved?
Yes. Autoclave at 121 °C for 15 minutes after dissolving the powder.
Is a positive result on Bile Esculin Agar confirmatory?
No. The result is presumptive. Some viridans streptococci, Listeria, Aerococcus, Leuconostoc and Pediococcus can also react, so confirm with tests such as growth in 6.5% NaCl and PYR.
Typical Formula (g/L)
Formula may be adjusted to meet performance requirements.
Test Procedure and Interpretation
Plates: Streak isolated colonies and incubate aerobically at 35–37 °C for 24–48 h. A dark brown to black zone around colonies is a positive reaction.
Slants: Inoculate the slant surface. Blackening of more than half of the slant within 48 h is positive. No blackening, or blackening of less than half, is negative (Facklam and Moody, 1970).
Confirmation: Confirm presumptive enterococci with additional tests, such as growth in 6.5% NaCl and the PYR test.
Related Standards (for user reference)
ISO 7899-2 (intestinal enterococci in water) uses the related Bile Aesculin Azide Agar for confirmation, which contains sodium azide. Bile Esculin Agar AS-1140 is the azide-free medium based on Swan (1954) and Facklam and Moody (1970).
References
- Swan A. The use of a bile-aesculin medium and of Maxted's technique of Lancefield grouping in the identification of enterococci (group D streptococci). J Clin Pathol. 1954;7:160–163.
- Facklam RR, Moody MD. Presumptive identification of group D streptococci: the bile-esculin test. Appl Microbiol. 1970;20(2):245–250.
- Facklam R. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin Microbiol Rev. 2002;15(4):613–630.
- Zimbro MJ, Power DA, et al., eds. Difco & BBL Manual: Manual of Microbiological Culture Media. 2nd ed. BD Diagnostics; 2009.
- ISO 7899-2:2000. Water quality — Detection and enumeration of intestinal enterococci — Part 2: Membrane filtration method.
Quality Control
Recommended Control Strains
Safety
Not classified as hazardous under the GHS. Not Dangerous Goods for transport. Avoid breathing dust and wear gloves and eye protection when weighing. Treat inoculated media as potentially infectious and autoclave before disposal. The full SDS is available for every lot.
Storage
Dehydrated medium: Store at 15–25 °C in a dry place, protected from light, with the container tightly closed. Use before the expiry date on the label.
Prepared medium: Store at 2–8 °C, protected from light. Validate the use period in your laboratory.