Staphylococcus Flashcards by Kyra Catedral

Specimens:

• Wound
• Blood
• Sputum
• Urine

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Gram staining

• Do direct gram stain from specimen
• Gram (+) cocci & neutrophils are
noted

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Staphylococcus: Culture Medium
Sheep’s Blood Agar Plate

BAP
SBA

• Medium, convex, creamy, dome shaped

• Pigment: white to golden yellow

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Colistin Nalidixic Acid Agar (CNA)

• Selective medium for growth of G(+) bacteria

• The medium is blood agar base with Antibiotics

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Growth on MSA

Type of media

Inhibitor

Indicator

• Selective & differential medium

• ↑ salt (7.5%) = selective

• Mannitol & phenol red

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Chromogenic Agar ( Hardy Chrome Staph aureus agar)
• Sau appears…
• S. epidermidis…
• S. saprophyticus…

deep pink to fuchsia colonies

partially or completely inhibited

appears turquoise colonies

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MACROSCOPIC CHARACTERISTICS
COLONIES
• size and texture
• margin
• color
•hemolysis

Medium to large smooth, butyrous,
creamy

Margin is entire

Staphyloxanthin (yellow pigment)

Narrow zone of ℬ-hemolysis (SBA)

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Test for Differentiation:
• Differentiate Staphylococcus from Streptococcus Test for

Catalase Test

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Catalase Test reagent

3% Hydrogen peroxide

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Test for Differentiation:

• Differentiate Staphylococcus
aureus from CoNS

Coagulase Test

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Coagulase Test reagent

Plasma (Fibrinogen) -> forms clot (+)

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Positive for coagulase indicates

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Slide coagulase

Tube coagulase

Bound coagulase
Free coagulase

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Test for Differentiation:

• Differentiate Micrococcus from Staphylococcus

Modified Oxidase Test

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Modified Oxidase Test Reagent

6% tetramethyl-p-phenylenediamine

dihydrochloride in dimethyl sulfoxide

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Modified oxidase

Results

• Result:
• (+) = dark blue - purple color
(Cytochrome C)
• (-)= no change in colo

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Test for Differentiation:

• Differentiate Micrococcus & Rothia
from Staphylococcus

Bacitracin Susceptibility Test

Bacitracin Susceptibility Test reagent

• 0.04 u Bacitracin disk

Bacitracin interpretation

Zones > 10 mm = Susceptible
Zones < 10 mm or no zone of inhibition
= Resistant

Test for Differentiation:

• Differentiate S. saprophyticus from other CoNS

Novobiocin Susceptibility Test

Results

• S. saprophyticus : Resistant
• other CoNS : Sensitive

Novobiocin susceptibility test reagent

• Place 5μg of Novobiocin

Novobiocin interpretation

• Susceptible = zone > than 16 mm
• Resistant = zone < or = to 16 m

Test for Differentiation:

• For the presumptive identification of S. aureus and differentiates it from CoNS.

DNAse Test

DNAse Test Results/ Interpretation

• Methyl Green: colorless • Toluidine Blue O: bright pink color. (Detection of thermostable nuclease)

DNAse Test Results/ Interpretation

• Methyl Green: colorless • Toluidine Blue O: bright pink color. (Detection of thermostable nuclease)

Members of the genus Staphylococcus are gram-positive spherical organisens about____ micrometer in diameter. They occur singly, in pairs, and in irregular clusters, and form yellow, orange, or white colonies on agar media.

1um

They are_____ tolerant and grow on ordinary bacteriological media as well as on the selective media used in this exercise.

salt

Usually, of the three Staphylococcus species of significance, S. aureus ferments mannitol, an alcoholic derivate of the hexose mannose, while S. epidermis and S. saprophyticus do not. Therefore, if the differential medium contains mannitol, the two species may be differentiated from one another. The salt concentration of mannitol salt agar is a high___% to inhibit other organisms, and mannitol is available for fermentation to acid. The indicator_____ is included in the medium to detect acid production. It will change from___ to ____ in the presence of acid-producing_______. No color change will occur with the other two species.

7% phenol red red to yellow S. aureus

S. aureus has the ability to produce_____, an enzyme that clots blood plasma and enhances its virulence by providing resistance to phagocytosis. The species also produces DNase to digest DNA and is sensitive to the antibiotic______. Of the three species, only______ is resistant to novobiocin.

coagulase novobiocin S. saprophyticus

In a clinical microbiology laboratory, a_____ swab specimen is routinely processed by inoculating onto (5).

nose BAP, Mac, CAP and thioglycollate or tryptic soy broth

When specimen contains Gram-positive cocci and yields(3), Staphylococcus species are suspected and these organisms are further isolated by plating onto____.

circular, opaque, smooth colonies MSA

Observe the plates for the presence of ***round opaque colonies that are white or pigmented.*** These are probably colonies of______

Staphylococcus species.

S. aureus produces ______pigmented colonies, while the other species produce white colonies. Also, colonies surrounded by a yellow halo contain bacteria that fermented the mannitol and probably contain____ Where no color change to yellow (red as neutral) has taken place, the colonies are possibly_____ or _____. Some strains of S. saprophyticus can ferment mannitol and produce acid. Another possibility is Micrococcus luteus.

golden-yellow S. aureus. S. epidermidis or S. saprophyticus

______ will be isolated on the medium (MSA). These bacteria also are gram-positive cocci but they occur in characteristic tetrads (clusters of four cells) or in cubical packets of eight cells (the sarcina configuration). The colonies on agar are bright yellow.

M. luteus

Catalase test reagent

3% hydrogen peroxide

Coagulase Prepare coagulase reagent, ______ amounts in 13 x 100 mm glass tubes. The tubes can be prepared in large numbers and refrigerated for 10 days, or frozen at -20°C for several months.

plasma with EDTA in 0.5 ml

COAGULASE Incubate the suspension for _____at _____ C and observe for the presence of a gel or clot that cannot be re-suspended by gentle shaking. A ropy, opaque precipitate in the plasma is not a clot. If no clot forms after 4 hours, the tube should be________. Rare isolates require such extended incubation. Some isolates may demonstrate a clot at 4 hours that subsequently lyses and is not detectable at the 24-hour inspection. Thus, all assays should be examined at _______ and reincubated if______.

1 to 4 hours 35°C to 37°C incubated at room temperature overnight 4 to 6 hours negative

COAGULASE Organisms that fail to clot the plasma within 24 hours are considered______ and must be identified by other methods.

coagulase negative

DNase Test Test for DNA digestion by flooding the plate with_______. DNA will normally react with it and form a very fine precipitate, which will give a cloudy appearance to the agar after several minutes. If DNA digestion has occurred, the area near the streak will remain____. The remainder of the plate, however, becomes cloudy. A dark background is recommended, and observations should be made from an angle to see the cloudiness. Compare your results with the control plate containing no bacteria.

1N hydrochloric acid (HCI) clear