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

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.