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 InterpretationResult Interpretation Presumptive IDPoor 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

Staphylococcus saprophyticus on MSA plate

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 luteus2.Staphylococcus aureus3.Staphylococcus

epidermidisWork as a team of two for the control organisms, then perform the skin swab

individually!

Day 1

Control Organisms Skin Inoculum

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

Day 2

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 InterpretationResult Interpretation SymbolBubbles Catalase is present +No bubbles Catalase is absent

Coagulase TestCoagulase activates fibrin and leads to clot formation Protective barrier around bacterial cells Virulence factor

Test determines presence of coagulase enzymeUsed for differentiating S. aureus from other Staphylococci.

Tube Coagulase Test

check no later than 24 hours after inoculation!

Tests for bound of free coagulase

Slide Coagulase TestTests for bound coagulasePositive if agglutination within 1 to 2 minutes

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