Bile Esculin Azide Agar

Bile Esculin Azide Agar

Highly Selective & Differential Medium for Isolation of Enterococci

Catalog Number: AS-1141

Overview

Bile Esculin Azide Agar is a highly selective and differential solid culture medium specifically designed for the isolation and presumptive identification of enterococci, including Enterococcus faecalis, E. faecium, E. durans, and E. avium, from clinical, food, water, and environmental samples.

The medium combines three selective agents—bile (oxgall), sodium azide, and salt—with the esculin–ferric citrate reaction system. This formulation provides maximum selectivity, allowing enterococci to grow while effectively inhibiting streptococci, staphylococci, Gram-negative bacteria, and most competing flora.

Bile Esculin Azide Agar is the official reference medium in ISO 7899-2 for the detection and enumeration of intestinal enterococci in water and is widely cited in food microbiology and public-health standards.

Key Features & Benefits

• Triple selective system for maximum suppression of background flora

• Strong inhibition of Gram-negative bacteria and non-enterococcal Gram-positives

• Esculin hydrolysis produces intense black halos around positive colonies

• Faster and cleaner results than Bile Esculin Agar alone

• Clear presumptive results typically visible within 18–24 hours

Typical Applications

Water & Environmental Microbiology

• Enumeration of enterococci in drinking, bathing, and wastewater

• Reference method for ISO 7899-2 compliance

Food Microbiology

• Detection of faecal enterococci in dairy, meat, and processed foods

• Hygiene monitoring and quality-control testing

Clinical & Research Laboratories

• Confirmation of enterococci from clinical specimens

• Supportive screening in antimicrobial-resistance workflows

Principle of the Medium

• Bile (oxgall) inhibits many Gram-positive bacteria

• Sodium azide suppresses cytochrome-containing organisms, especially Gram-negative bacteria

• Esculin is hydrolysed by enterococci to esculetin

• Ferric ammonium citrate reacts with esculetin to form a black iron complex

A positive reaction is indicated by black or dark-brown colonies surrounded by a black halo (≥5 mm).

Composition (Typical – per litre)

• Peptone: 17.0 g

• Beef extract: 3.0 g

• Yeast extract: 5.0 g

• Oxgall (bile): 10.0 g

• Esculin: 1.0 g

• Ferric ammonium citrate: 0.5 g

• Sodium chloride: 5.0 g

• Sodium azide: 0.15 g

• Agar: 15.0 g

Final pH at 25 °C: 7.1 ± 0.2

Preparation (Typical)

• Suspend 56.65 g of dehydrated medium in 1 L purified water

• Heat with frequent agitation and boil for 1 minute until fully dissolved

• Do not autoclave (esculin and azide are heat-sensitive)

• Cool to 45–50 °C, swirl gently, and pour into sterile Petri dishes

Incubation

• 35–37 °C for 24–48 hours

• Most enterococci show positive reactions within 24 hours

Interpretation

• Enterococci:
  Black or dark-brown colonies with a surrounding black halo

• Other organisms:
  No growth or growth without blackening

Storage & Stability

Dehydrated medium

• Store below 30 °C in a cool, dry place

Prepared plates

• Store at 2–8 °C, protected from light

• Use within 3 weeks

Safety Notice

This medium contains sodium azide, which is toxic and may form explosive compounds with heavy metals.
Handle with appropriate laboratory precautions and dispose of waste according to local regulations.

Intended Use

For laboratory clinical, food, water, environmental, and research microbiology use only.

Not for therapeutic use.

Quality & Compliance

• Official reference medium in ISO 7899-2

• Cited in APHA and FDA-BAM methods

• Manufactured under controlled conditions for batch consistency

• Certificate of Analysis (COA) available

• Safety Data Sheet (SDS) available upon request