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

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Activity 1.3: Physiological

The following tests may be performed on beta-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. Catalase Test
  2. Bacitracin Susceptibility
  3. CAMP Test
  4. SXT Susceptibility
  5. Sodium Hippurate Hydrolysis


1. Catalase Test

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide (3% solution), and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. Staphylococci produce catalase while streptococci do not.

Method

    Transfer some bacterial growth onto a glass slide. Add a drop of 3% hydrogen peroxide.

Results

    Positive = bubbles elaborated from bacteria

    Negative = no bubbling

    View catalase expected results.


2. Bacitracin Susceptibility

The growth of group A beta-hemolytic streptococci is inhibited by the antibiotic bacitracin. Other beta-hemolytic streptococci are not inhibited by bacitracin.

Method

    Streak a colony of beta-hemolytic streptococci for isolation on a sheep blood agar plate. With sterile forceps place a 0.04 U bacitracin disk onto the streaked plate in the second quadrant. Incubate the plate at 35°C overnight.

Results

    Positive = Sensitive = any zone of growth inhibition around the disk = group A streptococcus

    Negative = Resistant = no zone of inhibition around the disk

    View results patterns.


3. CAMP Test

Group B beta-streptococci produce a beta-hemolysin that reacts synergistically with the staphylococcal beta toxin produced by Staphylococcus aureus. Streptococci are inoculated perpendicularly to streaks of a beta toxin producing Staphylococcus aureus on sheep blood agar. The production of a distinct "arrowhead" of hemolysis indicates a positive test and is presumptive identification for group B streptococci.

Method

    Streak the beta-hemolytic Staphylococcus aureus in a straight line across the center of a sheep blood agar plate. At a right angle to (but not touching) the inoculum, streak the streptococcus to be tested. Incubate at 35°C overnight.

Results

    Positive = arrowhead of hemolysis at the junction of the two streaks = group B streptococcus

    Negative = no enhancement of hemolysis where the two meet

    View CAMP test reactions.


4. SXT Susceptibility

The growth of group C beta-hemolytic streptococci is inhibited by the antibiotic sulfamethoxazole-trimethoprim (SXT). Other beta-hemolytic streptococci are not inhibited by SXT.

Method

    Streak a colony of beta-hemolytic streptococci for isolation on a sheep blood agar plate. With sterile forceps place a SXT disk onto the streaked plate in the second quadrant. Incubate the plate at 35°C overnight.

Results

    Positive = Sensitive = any zone of growth inhibition around the disk = group C streptococcus

    Negative = Resistant = no zone of inhibition around the disk


5. Sodium Hippurate Hydrolysis

Group B streptococcus produce an enzyme called hippuricase, which other beta-hemolytic streptococci lack. This enzyme hydrolyzes sodium hippurate to two products, sodium benzoate and glycine. The presence of the enzyme is determined by detection of either of these end products.

Method

  1. Inoculate a 0.4 ml tube of 1% sodium hippurate very heavily.
  2. Incubate for 2 hours at 35°C.
  3. Add 0.3 ml of ninhydrin reagent.
  4. Incubate at 35°C for 10 minutes.
  5. Observe for the development of purple color.

Results

    Positive = purple color = group B streptococcus

    Negative = no color

    View results.