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15.2: Isolation and identification of Staphylococci

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    122719
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    1. Blood agar with a novobiocin (NB) disc

    To isolate staphylococci, clinical specimens are usually grown on Blood agar (described in Lab 14). Staphylococci produce round, raised, opaque colonies 1-2mm in diameter. The novobiocin disc is used to detect sensitivity or resistance to the antibiotic novobiocin.

    Test

    Fig. \(\PageIndex{1A}\): Staphylococcus aureus (pigmented strain) on Blood Agar

    Fig. \(\PageIndex{1B}\): Staphylococcus aureus (non-pigmented strain) on Blood Agar

    Fig. \(\PageIndex{1C}\): Staphylococcus epidermidis on Blood Agar Fig. \(\PageIndex{1D}\): Staphylococcus saprophyticuson Blood Agar
    Hemolysis (*) Usually beta(1) Usually gamma(2) Usually gamma(2)
    Pigment Often creamy gold(1) Usually white(2) Usually white(2)
    Novobiocin test Sensitive Sensitive Resistant

    (*) See Lab 14 for descriptions of hemolysis
    (1) some strains do not show hemolysis and/or pigment
    (2) some strains do show hemolysis and/or pigment
    sensitive = zone of inhibition around disc
    resistant = no zone of inhibition around disc

    Fig. \(\PageIndex{1A}\): Staphylococcus aureus (Pigmented Strain) Growing on Blood Agar

    Fig. \(\PageIndex{1B}\): Staphylococcus aureus (Non-pigmented Strain) Growing on Blood Agar

    Fig. \(\PageIndex{1C}\): Staphylococcus epidermidis Growing on Blood Agar

    Fig. \(\PageIndex{1D}\): Staphylococcus saprophyticus Growing on Blood Agar

    Photograph of <EM><em>Staphylococcus aureus</em></EM> growing on blood agar showing gold pigment, beta hemolysis and sensitivity to the novobiocin in the Taxo NB disk. Photograph of <EM><em>Staphylococcus aureus</em></EM> growing on blood agar showing no pigment and beta hemolysis Photograph of <EM>Staphylococcus epidermidis</EM> growing on blood agar showing no pigment, gamma reaction (no hemolysis) and sensitivity to the novobiocin in the Taxo NB disk. Photograph of <EM><em>Staphylococcus saprophyticus</em></EM> growing on blood agar showing no pigment, gamma reaction (no hemolysis) and resistance to the novobiocin in the Taxo NB disk.
    Note beta hemolysis (complete lysis of the red blood cells around the colonies; see arrows) on the blood agar and the organism is sensitive to the antibiotic novobiocin in the Taxo NB® disk. Note the non-pigmented colonies surrounded by a zone of beta hemolysis (complete lysis of the red blood cells around the colonies) on the blood agar. Note there is no hemolysis (gamma reaction) on the blood agar and the organism is sensitive to the antibiotic novobiocin as shown by the zone of inhibition around the Taxo NB® disk. Note there is no hemolysis (gamma reaction) on the blood agar and the organism is resistant to the antibiotic novobiocin in the Taxo NB® disk.

    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

    Image: Blood agar culture of Staphylococcus aureus © Rebecca Buxton, author.
    Licensed for use, ASM MicrobeLibrary.

    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

    2. Gram stain

    All staphylococci appear as Gram-positive cocci, usually in irregular, often grape-like clusters (see Fig. \(\PageIndex{1}\) above).

    3. Mannitol fermentation on Mannitol Salt agar (MSA)

    Staphylococci are able to tolerate the high salt concentration found in Mannitol Salt agar and thus grow readily. If mannitol is fermented, the acid produced turns the phenol red pH indicator from red (alkaline) to yellow (acid).

    Test Fig. \(\PageIndex{2A}\): Staphylococcus aureus Growing on Mannitol Salt Agar Fig. \(\PageIndex{2B}\): Staphylococcus epidermidis Growing on Mannitol Salt Agar Fig. \(\PageIndex{2C}\): Staphylococcus saprophyticus Growing on Mannitol Salt Agar
    Mannitol fermentation Positive Negative Usually positive

    positive = acid end products turn the phenol red pH indicator from red to yellow
    negative = prenol red remains red

    Fig. \(\PageIndex{2A}\): Staphylooccus aureus Growing on Mannitol Salt Agar.

    Fig. \(\PageIndex{2B}\): Staphylooccus epidermidis Growing on Mannitol Salt Agar.

    Fig. \(\PageIndex{2C}\): Staphylooccus saprophyticus Growing on Mannitol Salt Agar.

    Photograph of <EM>Staphylooccus aureus</EM> growing on mannitol salt agar and fermenting mannitol. Photograph of <EM>Staphylooccus epidermidis</EM> growing on mannitol salt agar and not fermenting mannitol. Photograph of <EM>Staphylooccus saprophyticus</EM> growing on mannitol salt agar and weakly fermenting mannitol.
    Mannitol salt agar is selective for staphylococci because of the high salt concentration. Acid from mannitol fermentation causes the pH indicator phenol red to turn from red (alkaline) to yellow (acid) Mannitol salt agar is selective for staphylococci because of the high salt concentration. There is no mannitol fermentation and, therefore, no acid production. The pH indicator phenol red remains red (alkaline) Mannitol salt agar is selective for staphylococci because of the high salt concentration. Acid from mannitol fermentation causes the pH indicator phenol red to turn from red (alkaline) to yellow (acid)
    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

    4. Production of coagulase

    The staphylococcal enzyme coagulase will cause inoculated citrated rabbit plasma to gel or coagulate. The coagulase converts soluble fibrinogen in the plasma into insoluble fibrin.

    Test Fig. \(\PageIndex{3A}\): Staphylococcus aureusCoagulase Test Fig. \(\PageIndex{3A}\): Staphylococcus epidermidisCoagulase Test Fig. \(\PageIndex{3C}\): Staphylococcus saprophyticusCoagulase Test
    Coagulase production Positive Negative Negative

    positive = plasma will gel or coagulate
    negative = plasma will not gel

    Fig. \(\PageIndex{3A}\): Positive Coagulase Test for Staphylococcus aureus

    Fig. \(\PageIndex{3B}\): Negative Coagulase Test for Staphylococcus epidermidis

    Fig. \(\PageIndex{3C}\): Negative Coagulase Test for Staphylococcus saprophyticus

    Photograph of a Positive Coagulase Test for <EM>Staphylococcus 
    aureus</EM> showing that the rabbit plasma has gelled. Photograph of a Negative Coagulase Test for <EM>Staphylococcus 
    epidermidis</EM> showing that the rabbit plasma has not gelled. Photograph of a Negative Coagulase Test for <EM>Staphylococcus 
    saprophyticus</EM> showing that the rabbit plasma has not gelled.

    Rabbit plasma has gelled indicating the bacterium is coagulase positive.

    Rabbit plasma has not gelled indicating the bacterium is coagulase negative.

    Rabbit plasma has not gelled indicating the bacterium is coagulase negative.
    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

    5. The Staphyloslide® Latex Test for cell-bound coagulase (clumping factor) and/or Protein A

    The Staphyloslide® Latex Test is an agglutination test that detects cell-bound coagulase (clumping factor) and/or Protein A. Approximately 97% of human strains of S. aureus possess both bound coagulase and extracellular coagulase. Approximately 95% of human strains of S. aureus possess Protein A on their cell surface (see Fig. 7). This test uses blue latex particles coated with human fibrinogen and the human antibody IgG. Mixing of the latex reagent with colonies of the suspected S. aureus having coagulase and/or Protein A bound to their surface causes agglutination of the latex particles.

    Test Fig. \(\PageIndex{4A}\): Staphyloside® Latex Test on Staphylococcus aureus Fig. \(\PageIndex{4B}\): Staphyloside® Latex Test on Staphylococcus epidermidis Fig. \(\PageIndex{4C}\): Staphyloside® Latex Test on Staphylococcus saprophyticus
    Cell-bound coagulase (clumping factor) and/or Protein A Positive Negative Negative

    positive = clumping of latex particles
    negative = no clumping of latex particles

    Fig. \(\PageIndex{4A}\): Staphyloslide® Test on Staphylococcus aureus

    Fig. \(\PageIndex{4B}\): Staphyloslide® Test on Staphylococcus epidermidis

    Fig. \(\PageIndex{4C}\): Staphyloslide® Test on Staphylococcus saprophyticus

    Photograph of a positive Staphyloslide® Test on <EM>Staphylococcus 
    aureus</EM> showing clumping of the latex particles. Photograph of a negative Staphyloslide® Test on <EM>Staphylococcus 
    epidermidis</EM> showing no clumping of the latex particles. Photograph of a negative Staphyloslide® Test on <EM>Staphylococcus 
    saprophyticus</EM> showing no clumping of the latex particles.

    The blue latex particles coated with human fibrinogen and human IgG have clumped together indicating the bacterium is positive for bound coagulase (clumping factor) and/or Protein A.

    The blue latex particles coated with human fibrinogen and human IgG have not clumped together indicating the bacterium is negative for bound coagulase (clumping factor) and/or Protein A.

    The blue latex particles coated with human fibrinogen and human IgG have not clumped together indicating the bacterium is negative for bound coagulase (clumping factor) and/or Protein A.

    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0
    Further Information \(\PageIndex{1}\)

    For further information on coagulase and Protein A associated with S. aureus, see the following CourseArc lessons:

    Staphylococci are also being identified using chemiluminescent labelled DNA probes complementary to species-specific bacterial ribosomal RNA (rRNA) sequences as well as by other direct DNA techniques.

    Contributors and Attributions

    • Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)


    This page titled 15.2: Isolation and identification of Staphylococci is shared under a not declared license and was authored, remixed, and/or curated by Gary Kaiser.

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