1. Ex. 14: Skin Cultures and Importance of Selective and Differential Media for Isolating Gram-Positive Cocci Objectives??

2. Table 14-2: Bacteria Commonly Found on Human Skin Bacterium Frequency of presence Propionibacterium spp.++ Staphylococcus epidermidis++ Staphylococcus aureus+ Micrococcus spp.+ Corynebacterium spp.++ Streptococcus pyogenes +/  ++ = nearly always present; + = commonly present; +/  = rare

3. MSA: Selective and Differential Medium for Gram + Cocci Composition: meat extract, casein, peptones; 7.5% NaCl, mannitol as only fermentable carbohydrate, phenol red indicator.

4. Table 14-1: Mannitol Salt Agar (MSA) Results and Interpretation ResultInterpretationPresumptive ID Poor growth or no growth (P) Organism is inhibited by NaCl Not Staphylococcus Good growth (G) Organism is not inhibited by NaCl Possible Staphylococcus Medium turns yellow (Y) Organism produces acid (A) from mannitol fermentation Possible pathogenic Staphylococcus aureus Medium remains red (R) Organism does not ferment mannitol. No reaction (NR) Nonpathogenic Staphylococcus

6. Staphylococcus saprophyticus on MSA plate

7. Materials needed per team of two students: Three MSA plates Two sterile cotton swab Two tubes with sterile saline Materials needed per table: Slant cultures of the following bacterial species: 1.Micrococcus luteus 2.Staphylococcus aureus 3.Staphylococcus epidermidis Work as a team of two for the control organisms, then perform the skin swab individually! Day 1

8. Control OrganismsSkin Inoculum

9. Materials needed per table:  Gram staining reagents  Hydrogen peroxide (3%), glass slides  Rabbit plasma vials for coagulase test Day 2

10. Catalase Test H 2 O 2 is by-product of aerobic respiration. Lethal to the cells.  Most aerobic organisms produce catalase for protection Catalase test differentiates among morphologically similar Gram+ cocci.

11. Table 14-2: Catalase Test Results and Interpretation ResultInterpretationSymbol BubblesCatalase is present+ No bubblesCatalase is absent­

12. Coagulase Test  Coagulase activates fibrin and leads to clot formation  Protective barrier around bacterial cells  Virulence factor  Test determines presence of coagulase enzyme  Used for differentiating S. aureus from other Staphylococci.

13. Tube Coagulase Test check no later than 24 hours after inoculation! Tests for bound of free coagulase

14. Slide Coagulase Test Tests for bound coagulase Positive if agglutination within 1 to 2 minutes

15. Table 14-5: Results of MSA Plates Colony Colony description Pigment Mannitol fermentation Gram stain Catalase reaction S. aureus S. epidermidis M. luteus Skin isolate #1 Skin isolate #2 Skin isolate #3