Bile Esculin Agar w/ Kanamycin

Intended Use:

Recommended for selective isolation and presumptive identification of bacteria of the Bacteroides fragilis group from mixed flora.

Composition**

Final pH (at 25°C): 7.1±0.2

**Formula adjusted, standardized to suit performance parameters # -Equivalent to beef extract φ -Equivalent to Oxgall

Directions

Suspend 44.6 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. DO NOT OVERHEAT.

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 (8). The unique ability of Enterococci to split esculin was reported by Meyer and Schonfeld (10). Enterococci and Group D Streptococci hydrolyse esculin to esculetin and dextrose, which reacts with ferric citrate producing brownish black precipitate (9). The use of esculin hydrolysis in identification of Enterococci was first cited by Rochaix (12). Bile Esculin Agar was originally formulated by Swan (13) for the isolation and identification of Group D Streptococci from food. Facklam and Moody (4,5) 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 (2) on the basis of esculin hydrolysis. However, other tests such as salt tolerance should be performed for identifying Enterococci (3).

Bile Esculin Agar with Kanamycin is recommended for the selective isolation and presumptive identification of Bacteroides fragilis group of bacteria from mixed flora. This medium is a modification of the original formulation of Swan (13). In this medium kanamycin is added to an enriched Bile Esculin Agar, enriched with hemin and vitamin K1. Hemin and vitamin K1 enriches and enhances the growth of Bacteroides species. Kanamycin selectively promotes the growth of Bacteroides fragilis while inhibiting the growth of facultative anaerobic and aerobic gram-negative bacilli. Anaerobes that are incapable of hydrolyzing esculin do not form brown or black pigmented colonies on this medium. The plates should be reduced by keeping in anaerobic jar for 18-24 hours, just before incubation (1).

Gelatin peptone and meat peptone B serves as sources of carbon, nitrogen, amino acids, vitamins and essential growth nutrients. Bile and kanamycin 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. Viridans Streptococci sometimes exhibit a weak positive reaction. Also, Leuconostoc, Pediococcus, Lactococcus species causing human infections give a positive bile esculin test (11). To enhance the growth of Enterococci, Bile Esculin Agar can be supplemented with 50ml/l horse serum (9).

Type of specimen

Isolated microorganism

Specimen Collection and Handling:

The test specimens can be directly streaked on the surface of the plate. The inoculated plates should be immediately incubated under anaerobic conditions at 35-37°C. Incubation should be carried out for upto 7 days. 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. Further biochemical and serological tests must be carried out for complete identification.

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 Light yellow 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 gel with a bluish tinge forms in Petri plates

Reaction Reaction of 4.46% w/v aqueous solution at 25°C. pH : 7.1±0.2

pH 6.90-7.30

Cultural Response

Cultural characteristics observed when incubated anaerobically, after an incubation at 35-37°C for 24-48 hours (in case of no growth incubation continued upto 7 days).

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 (6,7).

Reference

1. Dowell, 1975, Am. J. Med. Technol., 41:402.
2. Edberg S. C., Pittman S., and Singer J. M., 1977, J. Clin. Microbiol., 6:111.
3. Facklam R., 1973, Appl. Microbiol., 26:138.
4. Facklam R., 1972, Appl. Microbiol., 23:1131.
5. Facklam R. R and Moody M. D., 1970, Appl. Microbiol., 20(2):245.
6. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition
7. 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.
8. 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
9. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. I, Williams and Wilkins, Baltimore.
10. Meyer and Schonfeld, 1926, Zentralbl. Bakeriol, Parasitenk. Infectionskr. Hyg. Abt. Orig. 99:402.
11. 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.
12. Rochaix, 1924, Comt. Rend. Soc. Biol., 90:771.
13. Swan, 1954, J. Clin. Pathol., 7:160.

Revision: 03/2020