LSH4801
PORTFOLIO 2024
Unique Number: 569228
Due Date: 22 November 2024
QUESTION 1
1.1.
Immunofluorescence for Identifying Streptococcus pyogenes and Differentiating It
from Other Streptococci and Staphylococci
1. Application of Immunofluorescence to Identify Streptococcus pyogenes (Group A
Streptococcus)
Immunofluorescence is a laboratory technique used to detect specific antigens in a sample
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QUESTION 1
1.1.
Immunofluorescence for Identifying Streptococcus pyogenes and
Differentiating It from Other Streptococci and Staphylococci
1. Application of Immunofluorescence to Identify Streptococcus pyogenes
(Group A Streptococcus)
Immunofluorescence is a laboratory technique used to detect specific antigens in a
sample by using antibodies that are conjugated to a fluorescent dye. When exposed
to ultraviolet (UV) light, the dye emits fluorescence, allowing visualization of the
target antigen under a fluorescence microscope. This method can be highly effective
in identifying Streptococcus pyogenes, which is associated with a range of infections
such as pharyngitis, impetigo, and more severe systemic infections.
To identify S. pyogenes isolated from a patient sample using immunofluorescence,
the following steps would be taken:
1. Sample Preparation: The bacterial isolate, which has been cultured from the
patient sample, would first be prepared by creating a smear of the bacteria on
a glass microscope slide.
2. Application of Specific Antibodies: A primary antibody specific to
Streptococcus pyogenes would be applied to the bacterial smear. This
antibody is typically designed to recognize and bind to a specific antigen
found on the surface of S. pyogenes, such as the M protein or group A
carbohydrate antigen.
3. Incubation: The slide is incubated with the primary antibody, allowing it time
to bind to the bacterial antigens. After incubation, the slide is washed to
remove any unbound antibody.
4. Secondary Antibody with Fluorescent Dye: A secondary antibody, which is
conjugated to a fluorescent dye (such as fluorescein isothiocyanate, FITC),
would then be applied. The secondary antibody binds to the primary antibody.
The fluorescence emitted by the conjugated dye will allow visualization of the
bacteria under a fluorescence microscope.
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5. Visualization: The slide is examined under a fluorescence microscope. If S.
pyogenes is present, the bacteria will emit fluorescence, typically appearing
as bright green or yellow against a dark background. This confirms the
presence of S. pyogenes in the patient sample.
2. Differentiation of Streptococcus pyogenes from Other Morphologically
Similar Streptococci and Staphylococci
Differentiating Streptococcus pyogenes from other morphologically similar
streptococci (e.g., Streptococcus agalactiae or Streptococcus pneumoniae) and
staphylococci (e.g., Staphylococcus aureus) requires several biochemical and
microbiological tests in addition to immunofluorescence. Here's how this can be
achieved:
1. Gram Staining and Morphology:
o S. pyogenes is a Gram-positive cocci that appears as chains or pairs
under the microscope.
o Staphylococci (e.g., S. aureus) also appear as Gram-positive cocci but
are arranged in clusters (grape-like clusters), which distinguishes them
from the chain formation of S. pyogenes.
2. Hemolysis on Blood Agar:
o S. pyogenes produces beta-hemolysis (clear zone around the
colonies) on blood agar, indicating complete lysis of red blood cells due
to the production of streptolysin O and S toxins.
o Streptococcus agalactiae (Group B Streptococcus) also produces beta-
hemolysis but has different surface antigens, which can be identified
through further serological tests.
o S. pneumoniae produces alpha-hemolysis (greenish discoloration)
due to partial hemolysis of red blood cells from the action of
pneumolysin.
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o Staphylococcus aureus typically produces beta-hemolysis, similar to
S. pyogenes, but can be distinguished by other tests, such as
coagulase production.
3. Catalase Test:
o S. pyogenes is catalase-negative, meaning it does not produce the
enzyme catalase that breaks down hydrogen peroxide.
o Staphylococci, including S. aureus, are catalase-positive, which is a
key differentiating factor between streptococci and staphylococci.
4. Bacitracin Sensitivity Test:
o S. pyogenes is sensitive to bacitracin. A zone of inhibition will appear
around a bacitracin disk on a culture plate.
o Other streptococci, such as S. agalactiae, and S. pneumoniae are
generally resistant to bacitracin.
5. Lancefield Grouping:
o S. pyogenes is a Group A streptococcus, which can be identified using
Lancefield antigen grouping methods, where a specific reagent
identifies the presence of the Group A carbohydrate antigen.
o Other streptococci, such as S. agalactiae, belong to Group B, and S.
pneumoniae does not have a Lancefield antigen grouping.
6. Molecular Methods:
o PCR (Polymerase Chain Reaction) can be used to detect specific
genetic markers unique to S. pyogenes, such as the M protein gene or
SpeA toxin gene. This molecular technique can definitively
differentiate S. pyogenes from other similar organisms.
Conclusion
Immunofluorescence is a highly specific and rapid technique for identifying
Streptococcus pyogenes in clinical specimens. By using antibodies that target
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