Overview and Objectives
Main Topic
Subtopic 1:
Beta-hemolytic Streptococci
1.1, 1.2, 1.3
  Subtopic 1 Summary
Subtopic 2: Alpha-hemolytic streptococci
  Activity 2
  Subtopic 2 Summary
Subtopic 3: Gamma Streptococci
3.1, 3.2
  Subtopic 3 Summary
Module Summary

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Acvtivity 3.2: Physiological

The following tests may be performed on gamma-hemolytic streptococci for identification. All results should be recorded on the lab report. The student should draw a flowchart to indicate the identification scheme.

  1. Bile Esculin
  2. Salt Tolerance

1. Bile Esculin

Bile esculin incorporates two tests. Growth in the presence of 40% bile and the ability to hydrolyze esculin that combines with Fe +3 in the medium to form a black precipitate. Group D streptococci and Enterococcus are tolerant to bile and can hydrolyze esculin.


    Streak a loopful of streptococcal colony back and forth on the slanted surface of the bile esculin agar tube. Incubate at 35°C overnight.


    Bile tolerance

      Growth on the slant = positive

      No growth = negative

    Esculin hydrolysis

      Black precipitate = positive

      Growth on slant without black precipitate = negative

2. Salt Tolerance

Enterococcus are able to grow in the presence of 6.5% NaCl but group D streptococci cannot. This key trait distinguishes them from one another.


    Inoculate a tube of bain-heart infusion broth containing 6.5% NaCl lightly. Do not make turbidity evident. Incubate at 35°C overnight.


    Positive = growth/turbidity = group D Enterococcus

    Negative = no growth/clear broth = group D streptococci