Bile Esculin Azide Agar

Intended Use

For selective isolation and presumptive identification of faecal Streptococci.

Composition**

Final pH (at 25°C): 7.1±0.2

# Equivalent to Beef extract

## - Equivalent to Oxgall

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 56.65 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Caution: Sodium azide has a tendency to form explosive metal azides with plumbing materials. It is advisable to use enough water to flush off the disposables.

Principle And Interpretation

Group D Streptococci possess the group D lipoteichoic acid antigen in their cell walls. Former Group D species, which are predominant normal inhabitants of the human gastrointestinal tract, are termed as faecal Streptococci or Enterococci (1). The unique ability of Enterococci to split esculin was reported by Meyer and Schonfeld (2). Enterococci and Group D Streptococci hydrolyse esculin to esculetin and dextrose, which reacts with ferric citrate producing brownish black precipitate (3). The use of esculin hydrolysis in identification of Enterococci was first cited by Rochaix (4). Bile Esculin Agar was originally formulated by Swan (5) for the isolation and identification of Group D Streptococci from food. Facklam and Moody (6) further reported that using Bile Esculin Agar, Group D Streptococci could be differentiated from non Group D Streptococci.

Bile Esculin Agar was also shown to aid differentiation of Enterobacteriaceae, Klebsiella, Enterobacter, Serratia from other Enterobacteriaceae genera (7) on the basis of esculin hydrolysis. However, other tests such as salt tolerance should be performed for identifying Enterococci (8).

Bile Esculin Azide Agar is a modification of Bile Esculin Agar as per Isenberg (9). In this medium the bile concentration is reduced and additional sodium azide is incorporated.

Tryptone, proteose peptone and HM peptone B serves as sources of carbon, nitrogen, amino acids, vitamins and essential growth nutrients. Bile and sodium azide inhibits most of the other accompyning bacteria. Esculin in the medium is hydrolyzed to esculetin and dextrose. Esculetin reacts with ferric citrate to form a dark brown or black complex, visualized as a zone of black precipitate around the colonies. If the media is dispensed in tubes in the form of slants, a positive reaction is indicated by blackening of more than half of the slant within 24-48 hours. If blackening is totally absent or if less than half of the slant is blackened within 24-48 hours, the test is negative. Viridans Streptococci sometimes exhibit a weak positive reaction. Also, Leuconostoc, Pediococcus, Lactococcus species causing human infections give a positive bile esculin test (10). To enhance the growth of Enterococci, Bile Esculin Agar can be supplemented with 50ml/l horse serum (3). Suspected water samples are filtered using membrane filters. These membrane filters are aseptically placed on Slanetz and Bartely Medium (M612I). Red or maroon coloured colonies observed after incubation are further confirmed by aseptically transferring the membrane filter on to Bile Esculin Azide Agar plate preheated to 44°C. Incubation at 44 ± 0.5°C for 2 hours is done following the inoculation.

All typical colonies exhibiting a brown black colouration in the surrounding medium are counted as intestinal Enterococci (10). Alternatively Bile Esculin Azide Agar can also be used for direct isolation of Enterococci (without membrane filter), by incubation at 35-37°C for 18-24 hours.

Type of specimen

Clinical- Faeces, Food samples

Specimen Collection and Handling:

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (4).

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (8,9). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions :

In Vitro diagnostic use. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations :

1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user's unique requirement.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance
Cream to yellow homogeneous free flowing powder

Gelling
Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium
Amber coloured, clear to slightly opalescent gel with a bluish tinge forms in Petri plates.

Reaction
Reaction of 5.67% w/v aqueous solution at 25°C. pH : 7.1±0.2

pH
6.90-7.30

Cultural Response

Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.

Key: *Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (8,9).

Reference

1. Edberg S. C., Pittman S., and Singer J. M., 1977, J. Clin. Microbiol., 6:111.
2. Murray P. R., Baron E. J., Jorgensen J. H., Pfaller M. A., Yolken R. H.,(Eds.), 8th Ed., 2003, Manual of Clinical Microbiology, ASM, Washington, D.C.
3. Facklam R., 1972, Appl. Microbiol., 23:1131.
4. Salfinger Y., and Tortorello M.L., 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
5. Swan, 1954, J. Clin. Pathol., 7:160.
6. Facklam R. R and Moody M. D., 1970, Appl. Microbiol., 20(2):245.
7. Meyer and Schonfeld, 1926, Zentralbl. Bakeriol, Parasitenk. Infectionskr. Hyg. Abt. Orig. 99:402.
8. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
9. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
10. Rochaix, 1924, Comt. Rend. Soc. Biol., 90:771.