Ex. 14: Skin Cultures and Importance of Selective and Differential Media for Isolating Gram-Positive Cocci Objectives?? 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 MSA: Selective and Differential Medium for Gram + Cocci Composition: meat extract, casein, peptones; 7.5% NaCl, mannitol as only fermentable carbohydrate, phenol red indicator. Table 14-1: Mannitol Salt Agar (MSA) Results and Interpretation Result Interpretation Presumptive ID Poor growth or no Organism is inhibited Not Staphylococcus growth (P) by NaCl Organism is not Possible Good growth (G) inhibited by NaCl Staphylococcus Organism produces Medium turns yellow Possible pathogenic acid (A) from mannitol (Y) Staphylococcus aureus fermentation Organism does not Medium remains red Nonpathogenic ferment mannitol. No (R) Staphylococcus reaction (NR) Staphylococcus saprophyticus on MSA plate Day 1 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! Control Organisms Skin Inoculum Day 2 Materials needed per table: Gram staining reagents Hydrogen peroxide (3%) , glass slides Rabbit plasma vials for coagulase test Catalase Test H2O2 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. Table 14-2: Catalase Test Results and Interpretation Result Interpretation Symbol Bubbles Catalase is present + No bubbles Catalase is absent 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. Tube Coagulase Test Tests for bound of free coagulase check no later than 24 hours after inoculation! Slide Coagulase Test Tests for bound coagulase Positive if agglutination within 1 to 2 minutes Table 14-5: Results of MSA Plates Colony S. aureus S. epidermidis M. luteus Skin isolate #1 Skin isolate #2 Skin isolate #3 Colony description Pigment Mannitol fermentation Gram stain Catalase reaction