Purpose

 This test is used for the presumptive identification of enterococci and organisms in the Streptococcus bovis group. The test differentiates enterococci and group D streptococci from non–group D viridans streptococci.

Principle

 Gram-positive bacteria other than some streptococci and enterococci are inhibited by the bile salts in this medium. Organisms capable of growth in the presence of 4% bile and able to hydrolyze esculin to esculetin. Esculetin reacts with Fe³⁺ and forms a dark brown to black precipitate.

 Media: Beef extract (11 g), enzymatic digest of gelatin (34.5 g), esculin (1 g), ox bile (2 g), ferric ammonium citrate (0.5 g), agar (15 g), per 1000 mL, pH 6.6.

Method

1. Inoculate one to two colonies from an 18- to 24-hour culture onto the surface of the slant. 2. Incubate at 35°-37°C in ambient air for 48 hours.

Expected Results

Positive: Growth and blackening of the agar slant (Figure 1 A)

Negative: Growth and no blackening of medium (Figure 1, B)

No growth (not shown)

Limitations

As a result of nutritional requirements, some organisms may grow poorly or not at all on this medium.

Quality Control

Positive: Enterococcus faecalis (ATCC19433)— growth; black precipitate

Negative: Escherichia coli (ATCC25922)— growth; no color change

Streptococcus pyogenes (ATCC19615)—no growth; no color change

Fig1. Bile esculin agar. A, Positive. B, Negative.