Esculin Agar

Intended Use

Recommended for cultivation and differentiation of bacteria that can hydrolyze esculin and produce H₂S.

Composition**

Final pH (at 25°C): 7.3±0.2

**Formula adjusted, standardized to suit performance parameters # Equivalent to Beef heart infusion (solids)

Directions

Suspend 41.5 grams in 1000 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Distribute into screw-capped tubes in 3 ml volumes or as desired. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool tubes in a slanted position.

Principle And Interpretation

Esculin is a glycoside incorporated as a differential agent to facilitate the identification of various organisms, including Enterobacteriaceae, Enterococci and anaerobes.

Esculin Agar is based on the formula recommended (1), for the cultivation and differentiation of bacteria based on their ability to hydrolyze esculin and produce H₂S. The unhydrolyzed esculin can be detected using long wave UV light at 360 nm since they will remain unchanged and fluorescence under UV light (4,5). Hydrolyzed esculin will not fluoresce and medium turns black (5).

Tryptone and HM infusion solids B provide amino acids and other nitrogenous substances that support bacterial growth. Esculin is a differentiating agent, which helps in identification of esculin-positive organism. Esculin is hydrolyzed to dextrose and esculetin, which forms a brown black complex in the presence of iron salt (ferric citrate) (4). Blackening of the agar medium.

Type of specimen

Isolated Microorganism

Specimen Collection and Handling

For samples follow appropriate techniques for handling specimens as per established guidelines (2,3). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

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 specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Pure isolated colonies must be used for testing Esculin hydrolysis.

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 forms in tubes as slants

Reaction: Reation of 4.15% w/v aqueous solution at 25°C. pH: 7.3±0.2

pH: 7.10-7.50

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 sample must be decontaminated and disposed of in accordance with current laboratory techniques (2,3).

Reference

1. Atlas R. M., 1996, Handbook of Microbiological Media, 2nd Ed., CRC Press
2. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
3. 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.
4. Koneman E. W., Allen S. D., Janda W. M., Schreckenberger P. C., Winn W. C. Jr., 1997, Colour Atlas and Textbook of Diagnostic Microbiology, 5th Ed., J. B. Lippinccott- Raven Publishers, Philadelphia, Pa.
5. Shigei, 1992, In Isenberg (Ed.), Clinical Microbiology Procedures Handbook, Vol. 1, American Society for Microbiology, Washington, D.C.