Bile Esculin Agar Base

Intended Use

Recommended for differential isolation and presumptive identification of group D Streptococci in food and pharmaceutical products.

Composition**

Final pH (at 25°C): 6.6±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Meat extract

Directions

Suspend 31.75 grams in 500 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Cool to 45-50°C. Add rehydrated contents of 1 vial of Esculin (FD050). Mix and dispense into tubes or flasks as desired. Sterilize by autoclaving at 15lbs pressure (121°C) for 15 minutes. Allow the tubed medium to solidify in slanted position.

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 (7). The unique ability of Enterococci to split esculin was reported by Meyer and Schonfeld (9). Enterococci and Group D Streptococci hydrolyse esculin to esculetin and dextrose, which reacts with ferric citrate producing brownish black precipitate (8). The use of esculin hydrolysis in identification of Enterococci was first cited by Rochaix (11). Bile Esculin Agar was originally formulated by Swan (13) for the isolation and identification of Group D Streptococci from food. Facklam and Moody (2,3) 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 (1) on the basis of esculin hydrolysis. However, other tests such as salt tolerance should be performed for identifying Enterococci (4).

Bile Esculin Agar Base with added supplements is recommended for selective isolation and presumptive identification of group D streptococci from food and pharmaceutical products.

Peptone and HM peptone B serves as sources of carbon, nitrogen, amino acids, vitamins and essential growth nutrients. Bile inhibits most of the other accompyning bacteria. Esculin when added as a supplement 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 (8). Inoculate and incubate the test sample in Todd Hewitt Broth (M313). After 24 hours incubation add two drops of the culture onto the surface of slant or plate media (4,8).

Type of specimen

Food samples; Pharmaceutical samples.

Specimen Collection and Handling

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (12).

For pharmaceutical samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards.

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. Viridans Streptococci sometimes exhibit a weak positive reaction.

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 brownish 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 solution with a bluish tinge forms in Petri plates or in tubes as slants.

Reaction Reaction of 6.35% w/v aqueous solution at 25°C. pH : 6.6±0.2

pH 6.40-6.80

Cultural Response Cultural characteristics observed with added Esculin (FD050) in an increased atmosphere of Carbon dioxide, 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 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle inorder 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. Use before expiry date on the label.

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 (5,6).

References

1. Edberg S. C., Pittman S., and Singer J. M., 1977, J. Clin. Microbiol., 6:111.
2. Facklam R., 1972, Appl. Microbiol., 23:1131.
3. Facklam R. R and Moody M. D., 1970, Appl. Microbiol., 20(2):245.
4. Facklam R., 1973, Appl. Microbiol., 26:138.
5. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
6. 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.
7. Koneman E. W., Allen S. D., Janda W. M., Schreckenberger P. C., Winn W. C. Jr., 1992, Colour Atlas and Textbook of Diagnostic Microbiology, 4 th Ed., J. B. Lippinccott Company
8. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. I, Williams and Wilkins, Baltimore..
9. Meyer and Schonfeld, 1926, Zentralbl. Bakeriol, Parasitenk. Infectionskr. Hyg. Abt. Orig. 99:402.
10. 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.
11. Rochaix, 1924, Comt. Rend. Soc. Biol., 90:771.
12. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
13. Swan, 1954, J. Clin. Pathol., 7:160.