Gram Positive Cocci Flashcards
String Test
Reagent : 3% KOH Gram+/- Positive reaction= String formation Positive = Gram - Negative = Gram +
Gram-Sure: Remel
Reagent : L-alanine-7-amido-4-
methylcoumarin
Gram -/+
LanaGram: Hardy Diagnostics
Reagent : L-alanine-4-nitroanilide
Gram -/+
APNA; Key Scientific
Reagent : L-alanine-p-nitroanilide
Gram -/+
STAPHYLOCOCCUS AUREUS Clinical Importance
-Different Virulence factors, such as adhesins, enzymes, and
toxins.
-Resistant strains, most common cause of nosocomial
pneumonia & skin infections.
-S.aureus is second after CoNS as a cause of primary
bacteremia in hospitals.
-Persistent carriers (10-35%)
-Intermittent carriers (20-75%)
-Noncarriers (5-50%)
-(MRSA, MRSE)
Catalase Test
- 3% Hydrogen Peroxide
- Catalase Positive V. Negative
- Creation of bubbles
- Staphylococcus V. Streptococcus
- Staph = Positive
- Strept = Negative
STAPHYLOCOCCUS:
General Characteristics
Gram-positive cocci in pairs, tetrads, and
clusters
-Catalase positive
- Most species are halotolerant: resistant to
10% NaCl
-Susceptible to lysostaphin, resistant to
lysozyme
STAPHYLOCOCCUS Aureus : Presumptive Positive
-Catalase positive
-Gram-positive cocci in clusters
-Tube or slide coagulase or latex
agglutination test positive
Slide Coagulase Test
Clumping Factor: Demonstrated by the ability of the organism to act directly on the fibrinogen in the plasma to clump it in a slide assay. -Rabbit Plasma EDTA- -S. Aureus= Coag + if Negative move to tube test
Tube Coagulase Test-Rabbit
Plasma Method
Tube Coagulase:
Coagulase is a thermostable thrombin-like
substance that activates fibrinogen to form fibrin,
resulting in a fibrin clot. This is demonstrated in
the test tube by the formation of clot when rabbit
plasma is inoculated with the Staphylococcus for 4
to 24 hrs.
The coagulase clot can be destroyed by S.aureus
fibrinolysin or staphylokinase, an enzyme which is
more active at 35°C.
-Rabbit Plasma EDTA-
-S. Aureus= Coag +
Coagulase Test-Protein A/
Clumping Factor Agglutination
Method
Staphylococcal protein A (SAG) or clumping
factor agglutination:
S. aureus produces another substance in its cell
wall, protein A, which binds to the FC part of
IgG. If latex coated with IgG and with human
fibrinogen, a Staphylococcus will agglutinate if
either clumping factor or protein A is present in
the bacterial cell wall.**
-S. Aureus= Coag +
Voges–Proskauer (VP Test)
-Voges–Proskauer: VP is a test used to
detect acetoin in a bacterial broth culture.
-The test is performed by adding alpha-
naphthol and 40% potassium hydroxide to culture if acetylmethyl carbinol(acetoin) is present then it turns red.
-Incubate 24hr take 2mls added reagents and mix to check for color change.
-Voges-Proskauer broth which has been
inoculated with bacteria. A cherry red color
indicates a positive result, while a yellow-
brown color indicates a negative result.
-All staphylococci except, S. intermedius
and S. hyicus, are positive for VP.
PYR Test
- L-pyroglutamic acid beta-
naphthylamide
-pyrolidonyl arylamidase
-PYR test is a rapid colorimetric method for
presumptive identification of certain groups of
bacteria based on the activity of the enzyme
pyrolidonyl arylamidase. L-pyroglutamic acid beta-
naphthylamide is impregnated into the test disk
and serves as the substrate for the detection of
pyrolidonyl arylamidase. Hydrolysis of the
substrate yields beta-naphthylamide which
combines with the PYR Reagent (p-dimethylamino-
cinnamaldehyde) to form a bright pink to cherry
red color within 2 minutes. - PYR activity is a key
test for differentiation of some species of
coagulase-negative Staphylococcus.
Ornithine Decarboxylase
- The purpose is to see if the microbe can use the amino acid ornithine as a source of carbon and energy for growth. Use of ornithine is accomplished by the enzyme ornithine decarboxylase.
- A medium containing ornithine and a pH indicator is used. When ornithine is used, the pH of the medium rises and the indicator changes color.
- The medium used is ornithine decarboxylase broth. The medium is a nutrient broth to which 0.5% ornithine is added. An important component of the medium is a modest amount of glucose, necessary for the process to proceed. The pH indicator brom cresol purple is purple at neutral or alkaline/basic pH but turns yellow at pH <5.2.
- An inoculum from a pure culture is transferred aseptically to a sterile tube of ornithine decarboxylase broth. The inoculated tube is incubated at 35-37 C for 24 hours and the preliminary results are determined. The microbe must first use the glucose present to cause the pH to drop. This is indicated by a change from purple to yellow. Once the medium has been acidified, the enzyme ornithine decarboxylase is activated. The culture is incubated an additional 24 hours at 35-37 C to allow the microbe to now use the ornithine. The final results are then obtained by observing the tube at 48 hours. Change back to purple from yellow indicates a positive test for ornithine decarboxylase. Failure to turn yellow at 24 hours or to revert back to purple at 48 hours indicates a negative result.
- Differentiation of S. Lugdensis
- Arginine decarboxylase is useful in the identification of Enterococcus to the species level; Enterococcus faecalis and Enterococcus faecium are arginine positive but, Enterococcus avium is arginine negative.
Staphylococcus saprophyticus
Catalase positive Coagulase negative Non hemolytic Novobiocin resistant (<16mm) UTI -Polymyxin B sensitive Other novobiocin-resistant species: S.cohnii S.kloosii S.xylosus
