Bile Esculin Agar
Intended use
Recommended for isolation and presumptive identification of group D Streptococci from food and pharmaceutical
products.
Composition**
Ingredients Gms / Litre
Peptone 5.000
HM peptone B # 3.000
Bile □ 40.000
Esculin 1.000
Ferric citrate 0.500
Agar 15.000
Final pH ( at 25°C) 6.6±0.2
Directions
Suspend 64.5 grams in 1000 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Mix and dispense
into tubes or flasks as desired. Sterilize by autoclaving at 15 lbs 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 (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 (4) for the isolation and identification of Group D Streptococci from food. Facklam and
Moody (2,5) 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 (11) on the basis of esculin hydrolysis. However, other tests such as salt tolerance should
be performed for identifying Enterococci (3).
The medium is highly nutritious. Peptone and HM peptone B serves as sources of carbon, nitrogen, amino acids, vitamins
and essential growth nutrients. Bile inhibits most of the other accompanying 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. Viridians Streptococci sometimes exhibit a weak positive
reaction. Also, Leuconostoc, Pediococcus, Lactococcus species causing human infections give a positive bile esculin test
(6). To enhance the growth of Enterococci, Bile Esculin Agar can be supplemented with 50ml/L horse
serum (9). 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 (3, 9).
Type of specimen
Food samples
Specimen Collection and Handling:
Please refer disclaimer Overleaf.
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. This medium is general purpose medium and may not support the growth of fastidious organisms.
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 or in tubes as slants.
Reaction
Reaction of 6.5% w/v aqueous solution at 25°C. pH : 6.6±0.2
pH
6.40-6.80
Cultural Response
Cultural characteristics observed in an increased atmosphere of Carbon dioxide after an incubation at 35-37°C for 18-24
hours.
Organism Inoculum
(CFU)
Growth Recovery Esculin
Hydrolysis
Enterococcus faecalis ATCC
29212 (00087*)
50-100 luxuriant =50% positive
reaction,blackening
of medium
around the
colony
Proteus mirabilis ATCC
25933
50-100 luxuriant =50% negative
reaction
Streptococcus pyogenes
ATCC 19615
50-100 none-poor =10% negative
reaction
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).
