Dehydrated culture media
Bile Esculin Azide Agar ISO
For the selective isolation and presumptive identification of intestinal enterococci by membrane filtration method.
- Cat. No.
- LC-1005
- Category
- Dehydrated culture media
- Format
- 500 g
- Storage
- 2 to 25 °C
- Minimum guaranteed shelf life
- 15 months
- Standards
- ISO 7899-2
- Also available
- Ready-to-use medium, 1 format (LC-952)
- Also available
- Dehydrated medium, 1 format (LC-1372)
- Also available
- Ready-to-use medium, 1 format (LC-4701)
Bile Esculin Azide Agar is a modification of Bile Esculin Agar (LC-1031), with the addition of sodium azide as an inhibitor and with the reduction of the bile concentration. The resulting medium is more selective but still provides rapid growth and efficient recovery of enterococci. The ability to hydrolyze esculin in the presence of bile is a characteristic of enterococci. Organisms positive for esculin hydrolysis hydrolyze the glycoside esculin to esculetin and dextrose. The esculetin reacts with the ferric ammonium citrate to form a dark brown or black colony. Ox bile does not inhibit enterococci while other Gram-positive bacteria are inhibited. Sodium azide inhibits Gram-negative bacteria. Tryptone, peptone and yeast extract supply the nutrients essential for growth. Sodium chloride provides the osmotic balance. Bacteriological agar is the solidifying agent.
Key features
Applications
Selective isolation of enterococci.
Industry
Water.
Standards
ISO 7899-2.
Preparation
56.6 g in one liter of distilled water; boil for one minute, sterilize in autoclave at 121 °C for 15 minutes.
Principles and uses
The presence of intestinal enterococci is an indicator for faecal contamination, especially when the contamination occurred a long time before and the less resistant coliform bacteria, including Escherichia coli, may already be dead when the analysis is carried out.
Tolerance to bile and the ability to hydrolyze esculin constitute a reliable presumptive test for the identification of enterococci. A brown color (positive reaction) appears around the colonies.
Technical specifications
| Solubility | Without residues |
|---|---|
| Appearance | Fine powder |
| Color of the dehydrated medium | Toasted |
| Color of the prepared medium | Litmus |
| Final pH (25 °C) | 7.1 ± 0.1 |
| Amount per litre | 56.6 g |
| Storage | 2 to 25 °C |
Formula
| Ingredient | g/L |
|---|---|
| Bacteriological agar | 15 |
| Esculin | 1 |
| Ferric ammonium citrate | 0.5 |
| Ox bile | 10 |
| Peptone | 3 |
| Sodium azide | 0.15 |
| Sodium chloride | 5 |
| Tryptone | 17 |
| Yeast extract | 5 |
Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.
Preparation and use
Preparation
Suspend 56.6 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121 °C for 15 minutes. Cool to 50-60 °C and dispense into appropriate containers. Overheating can cause darkening of the medium. If tubes are used, allow cooling in a slanted position.
According to ISO 7899-2
For the detection and enumeration of enterococci according to ISO 7899-2:
- Filter a measured volume of water through a membrane filter.
- Place the membrane on a Slanetz-Bartley Medium (LC-1109).
- Incubate at 36 ± 2 °C for 44 ± 4 h.
- Transfer the membrane with characteristic colonies previously incubated in the Slanetz-Bartley medium (LC-1109), without inverting the membrane, to a plate with Bile Esculin Azide Agar, pre-heated to 44 °C.
- Incubate at 44 ± 0.5 °C for 2 hours.
- Read the plate immediately.
- It is considered that the typical colonies that show a brown-black color in the surrounding medium give positive reactions and are counted as intestinal enterococci.
Quality control
Microbiological test
Incubation conditions: 44 ± 0.5 °C / 2 h.
Inoculation conditions: confirmation (transference from membrane previously incubated in Slanetz-Bartley medium [E. faecalis and E. faecium] / TSA [E. coli]).
| Microorganism | Characteristic reaction |
|---|---|
| Enterococcus faecalisATCC 19433 | Brown-black halo |
| Escherichia coliATCC 25922 | Absence of brown-black halo |
| Enterococcus faecalisATCC 29212 | Brown-black halo |
| Enterococcus faeciumATCC 6057 | Brown-black halo |
Bibliography
- ISO 7899-2. Water quality - Detection and enumeration of intestinal enterococci - Part 2: Membrane filtration method.
- Facklam, R.R. and M.D. Moody 1970. Presumptive identification of Group D Streptococci: The bile-esculin test. Appl. Microbiol 20:245.
- Ruoff, K.L. 1995 Streptococcus. In P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H. Yolken (eds), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
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