Expected Cultural Response: Cultural response on Blood Agar Base, Improved with 5% sheep blood at 35 ± 2C after 18 - 24 hours incubation.
The organisms listed are the minimum that should be used for quality control testing.
Test Procedure
1. Process each specimen as appropriate, inoculate directly onto the surface of the medium. Streak for isolation with inoculating loop, stab agar several times to deposit beta-hemolytic streptococci beneath agar surface. Subsurface growth will display the most reliable hemolytic reactions owing to activity of both oxygen-stable and oxygen-labile streptolysins.6
2. Incubate plates aerobically, anaerobically, or under conditions of increased CO2 (5 - 10%) in accordance with established laboratory procedures.
Results
Examine medium for growth and hemolytic reactions after 18 - 24 and 48 hours incubation. There are four types of hemolysis on blood agar media described as:7
1. Alpha hemolysis () is the reduction of hemoglobin to methemoglobin in the medium surrounding the
colony. This produces a green discoloration of the medium.
2. Beta hemolysis () is the lysis of red blood cells, producing a clear zone surrounding the colony.
3. Gamma hemolysis (γ) indicates no hemolysis. No destruction of red blood cells occurs and there is no
change in the medium.
4. Alpha-prime hemolysis (ά) is a small zone of complete hemolysis surrounded by an area of partial lysis.
Storage
Store sealed bottle containing the dehydrated medium at 2 - 30°C. Once opened and recapped, place container in a low humidity environment at the same storage temperature. Protect from moisture and light by keeping container tightly closed.
Expiration
Refer to expiration date stamped on the container. The dehydrated medium should be discarded if not free flowing, or if appearance has changed from the original color. Expiry applies to medium in its intact container when stored as directed.
Limitations of the Procedure
1. Hemolytic reactions of some strains of group D streptococci have been shown to be affected by differences in animal blood. Such strains are beta-hemolytic on horse, human, and rabbit blood agar and alpha-hemolytic on sheep blood agar.6
2. Incubation atmosphere can influence hemolytic reactions of beta-hemolytic streptococci.6 For optimal performance, incubate blood agar base media under increased CO2 (5 - 10%).
Microorganism
Approx. Inoculum (CFU)
Response
Reactions
Escherichia coli ATCC® 25922
10 - 300
Fair to excellent growth
Beta hemolysis
Staphylococcus aureus ATCC® 25923
10 - 300
Fair to excellent growth
Beta hemolysis
Streptococcus pneumoniae ATCC® 6305
10 - 300
Fair to excellent growth
Alpha hemolysis
Streptococcus pyogenes ATCC® 19615
10 - 300
Fair to excellent growth
Beta hemolysis