Staphylococcus Flashcards
Specimens:
• Wound
• Blood
• Sputum
• Urine
Gram staining
• Do direct gram stain from specimen
• Gram (+) cocci & neutrophils are
noted
Staphylococcus: Culture Medium
Sheep’s Blood Agar Plate
BAP
SBA
• Medium, convex, creamy, dome shaped
• Pigment: white to golden yellow
Colistin Nalidixic Acid Agar (CNA)
• Selective medium for growth of G(+) bacteria
• The medium is blood agar base with Antibiotics
Growth on MSA
Type of media
Inhibitor
Indicator
• Selective & differential medium
• ↑ salt (7.5%) = selective
• Mannitol & phenol red
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
SAU
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)
Test for Differentiation:
• Differentiate Staphylococcus from Streptococcus Test for
Catalase Test
Catalase Test reagent
3% Hydrogen peroxide
Test for Differentiation:
• Differentiate Staphylococcus
aureus from CoNS
Coagulase Test
Coagulase Test reagent
Plasma (Fibrinogen) -> forms clot (+)
Positive for coagulase indicates
SAU
Slide coagulase
Tube coagulase
Bound coagulase
Free coagulase
Test for Differentiation:
• Differentiate Micrococcus from Staphylococcus
Modified Oxidase Test
Modified Oxidase Test Reagent
6% tetramethyl-p-phenylenediamine
dihydrochloride in dimethyl sulfoxide
Modified oxidase
Results
• Result:
• (+) = dark blue - purple color
(Cytochrome C)
• (-)= no change in colo
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
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
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
