Ex. 13: Selective Media for Isolating Gram

Report
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

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