There are three main types of hemolysis seen on sheep blood agar (SBA) plates: alpha, beta, and gamma.

Alpha hemolysis:
Alpha hemolysis will cause a greening of the agar. This is due to the partial breakdown of the surrounding red cells by the bacteria. In real practice, this sometimes can be difficult to see.

Beta hemolysis:
Beta hemolysis causes complete lysis of red cells surrounding the bacterial colonies. In Strep pyogenes, beta hemolysis is caused by the enzyme streptolysin. Other strains of bacteria including Staph aureus contain hemolytic properties.

Gamma hemolysis:
This means no hemolysis. Don’t get tricked by this one!

PYR Test:
I’m glad PYR has an abbreviation because it stands for L-pyrrolidonyl-beta-naphthylamide. The enzyme L-pyrroglutamyl-aminopeptidase, which is present in Strep pyogenes, hydrolyzes PYR to create beta-naphthylamine. (Insert graphic) When the indicator cinnamaldehyde is added you will get a bright red color if beta-naphthylamine is present. If not you will have no color change.

Hippurate Test:
The enzyme hippuricase is in many bacteria. If present, it will hydrolyze hippurate to produce glycine. The indicator ninhydrin will react with glycine to produce a dark purple color. No reaction will have no color change.

CAMP Test:
Some bacteria produce CAMP factor, an extracellular protein that acts synergistically with beta-lysin of Staph aureus to enhance red cell destruction leading to a larger zone of hemolysis. The test is setup by streaking a straight line of Staph aureus down the center of a SBA plate. Then, sample organism is inoculated in a line at a right angle to the Staph aureus, it should be close to the line but not touching. The plate is incubated overnight at 35C in O₂. A positive CAMP test will form a large area of hemolysis that looks like an arrow. A negative test will give no unique enhanced zone.

Nerdy Note
The test is named CAMP after the people who created the test (Christie, Atkins, Munch-Peterson).

Bile Esculin Agar Test:
Bacteria that can survive in up to 40% bile and hydrolyze esculin (sounds terrible right?) will convert ferric ammonium citrate to a dark brown or black from yellow. (insert graphic)

6.5% NaCl Test:
Bacteria are challenged to survive in a 6.5% NaCl broth solution.

Perspective Note
The oceans salt content is about 3.5%

Optochin Disc Test:
Optochin is a derivative of hydroquinine which was used to treat pneumococcal infections in the early 1900’s. It is primarily used to diagnose Strep pneumoniae.

Some conditions and other notes associated with some Strep species and G+ cocci:

Strep pneumoniae:
Associated with pneumonia, otitis media (ear infection), and adult meningitis. Tests positive for bile solubility and quellung tests.

Strep viridans:
Associated with dental caries, aka cavities.

Strep pyogenes (Group A):
Associated with Strep throat, scarlet fever, glomerulonephritis, impetigo, and rheumatic fever. Presence of Anti-streptolysin O (antibody produced in response to Strep pyogenes) can be clinically significant.

Strep agalactiae (Group B):
Associated with newborn meningitis

Phenylethyl alcohol agar (PEA):
Selective for G+ cocci

Streptolysin O and S:
Both hemolysins produced by Strep spp cause beta-hemolysis on SBA. S is more effective on the surface of the plate, O is more effective when the culture is stab inoculated because O is more sensitive to oxygen.