While we have learned how to do several biochemical tests so far there are a plethora of other tests that can be done. Here we include a couple other important presumptive tests.
*These tests have been done for you but you will still be responsible for understanding the basis and how to read these tests. You may need these later in the semester when we work with unknown bacteria.
CAMP factor is a diffusible, heat-stable protein produced by group B streptococci. This is a synergistic test between Staphylococcus aureus and Streptococcus agalactiae. S. agalactiae produces CAMP factor. S. aureus produces sphingomyelin C, which binds to red blood cell membranes. The two bacteria are streaked at 90o angles of one another. They do NOT touch. The CAMP factor produced by S. agalactiae enhances the beta-hemolysis of S. aureus by binding to already damaged red blood cells. As a result, an arrow of beta-hemolysis is produced between the two streaks. The test is presumptive for S. agalactiae that produces CAMP factor.
In the picture here, Streptococcus agalactiae was streaked throughout the top region of the plate and brought down toward the center of the plate. Staphylococcus aureus was streaked in a straight line across the center of the plate. Rings of hemolysis are evident all around S. aureus, however the hemolysis if greatly enhanced (in an arrow shape) where the S. agalactiae crosses the hemolysis rings.
This is a medium that is both selective and differential. It tests the ability of organisms to hydrolyze esculin in the presence of bile. It is commonly used to identify members of the genus Enterococcus (E faecalis and E. faecium).
The first selective ingredient in this agar is bile, which inhibits the growth of Gram-positives other than enterococci and some streptococci species. The second selective ingredient is sodium azide. This chemical inhibits the growth of Gram-negatives.
The differential ingredient is esculin. If an organism can hydrolyze esculin in the presence of bile, the product esculetin is formed. Esculetin reacts with ferric citrate (in the medium), forming a phenolic iron complex which turns the entire slant dark brown to black. The tube on the far right was inoculated with E. faecalis (positive). The tube in the center was inoculated with a bile esculin negative organism and the tube on the left was uninoculated.
This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus mirabilis is a rapid hydrolyzer of urea (center tube pictured here). The tube on the far right was inoculated with a urease negative organism and the tube on the far left was uninoculated.
