Analysis of Practical Microbiology Data
Medical Microbiology – Identification of Streptococcus spp.
Name:
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Objective:
The genus, Streptococcus, includes a number of pathogens of concern to human health. The
species in this genus are Gram Positive cocci. Microscopic observation reveals that these species
exist is chains or pairs. Streptococcus pyogenes is the causative agent of a number of diseases,
including strep throat, scarlet fever, and rheumatic fever. Streptococcus pneumoniae is the most
common cause of bacterial pneumonia. Streptococcus mutans is the causative agent of dental
caries, or cavities. These three species are significant pathogens for both males and females.
Streptococcus agalactiae, also referred to as Group B Streptococcus, is an important pathogen
during pregnancy and is one of the leading causes of infant mortality during the first few days
following birth.
Given that these species have a similar if not identical appearance with a Gram stain, it is
important to distinguish these organisms from each other and from other pathogens and normal
biota. The objective of this data analysis exercise is to demonstrate the diagnostic tools that are
used for differentiation of pathogenic streptococci, and to provide practice in interpretation of the
results from these diagnostic tests.
Learning Outcomes:
1. To understand the breadth of the pathogenic streptococci significant to human health
2. To become familiar with the various diagnostic tools that allow us to differentiate the
pathogens in this genus
3. To evaluate and interpret diagnostic tests for this group
1.
Connect to the following video file to observe the process for this exercise.
https://www.youtube.com/watch?v=e1UPNfi5VxY
2.
One of the first tests we perform after the Gram stain is to culture the
bacterium of sheep blood agar (SBA). SBA is made by preparing a agar medium
base, autoclaving it, cooling down to about 50ºC, then adding fresh sheep blood to
the medium. Once solidified, SBA has an opaque red appearance. SBA support a
wide range of human pathogens and is often the first medium used to cultivate
human clinical samples.
Different bacteria produce enzymes that break down red blood cells. Depending
on the enzymes produced degradation of the blood cells may either be partial,
complete, or non-detectable. The figure below illustrates these degrees of
degradation. α-hemolysis is a partial degradation of the blood cells, resulting in a
greenish color to the SBA directly under the growth of the organism. β-hemolysis
results in complete hydrolysis of the blood cells, creating a clear halo around the
areas of growth. When there is no detectable degradation of the red blood cells
directly under growth on the plate we refer to the reaction as γ-hemolysis.
A - β-hemolysis
B - α-hemolysis
C - γ-hemolysis
α-hemolysis
β-hemolysis
γ-hemolysis
3.
Record your observation of the three types of hemolysis in the table
below.
Streptococci on Sheep Blood Agar
Hemolysis Type
Observations/Interpretations
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α
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β
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γ
4. Look at the unknown streptococci below. Determine the hemolysis class for
each.
Unknown 1
Unknown 2
Unknown 3
5.
Record the characteristics of each unknown in the table that follows.
Hemolytic Characteristics of Unknowns
Unknown
Observations and Interpretations
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1
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2
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3
6.
Next observe the Gram stains for the three unknowns that follow. Record
your observations and interpretations in the subsequent table.
Unknown 1
Unknown 2
Unknown 3
Gram Stains of Unknowns
Unknown
Observations and Interpretations
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1
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2
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3
7.
There are certain tests you can perform to more accurately determine the
species of Streptococcus you are analyzing. One of these is the CAMP test.
CAMP factor is produced and secreted by certain bacteria, including S. agalactiae.
When CAMP is secreted in proximity to Staphylococcus aureus CAMP amplifies
the β-hemolysis of S. aureus. When strains of strep are streaked at a right angle to
a line of inoculum of the staph, an arrowhead shaped area of hemolysis is seen at
the end of the streak of the CAMP positive strain that is proximal to the staph.
Positive CAMP Test
8.
You test your unknown strains with the CAMP test. The results are shown
in the following image.
Can you draw any conclusions at
this point? Why or why not? If so,
what conclusions can you draw?
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9.
Based on your hemolysis results you should now know that you have one
α-hemolytic isolate and two β-hemolytic isolates. There are pathogens of concern
in each group, but there are also streptococci in these classes that are either nonpathogenic, or only opportunistic pathogens. There are two disk diffusion tests we
can apply to determine if primary pathogens are present. To test α-hemolytic strep
we use a chemical called optochin. Within the α-hemolytic group, Streptococcus
pneumoniae is sensitive to optochin while most of the other streps in this group are
resistant. To determine if your α-hemolytic isolate is S. pneumoniae you streak the
suspect isolate on a blood agar plate and drop an optochin disk onto the first streak
quadrant (heaviest area for growth). After incubation you observe the following.
What are your observations of this
test and what can you conclude?
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10.
There is also a disk diffusion test for your β-hemolytic isolates.
Bacitracin is an antibiotic that can be used to differentiate species in this
hemolytic group. Among the β-hemolytic streps there are a number of pathogenic
strains. One of the most serious of these is S. pyogenes. This is the causative agent
of strep throat and, depending on the strain, scarlet fever and rheumatic fever. S.
pyogenes is sensitive to bacitracin. Other β-hemolytic streps also include
pathogens, including Streptococcus agalactiae, an extremely virulent species for
newborns that is the leading cause of infant mortality during the first month
following birth. There are other species in the β-hemolytic group, but the two
mentioned above are the most serious pathogens. You test your two β-hemolytic
isolates for sensitivity to bacitracin. The results follow.
Describe what you see in the figure to the left.
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11.
The final test we will analyze is
that developed by Rebecca Lancefield and
is referred to as the Lancefield Typing
method. Rebecca Lancefield developed
antibodies that are specific to different
carbohydrates on the surfaces of
streptococci. These antibodies designate
streps into serogroups called Groups A-S.
Some streps do not contain these carbohydrate antigens and as a result are nonLancefield Group streptococci. The antibodies are attached to latex beads so that
when the antibodies react with a particular antigen the latex beads clump and form
visible grains on a slide. An example of a latex bead immunoagglutination
reaction is shown to the right.
12. You test your two β-hemolytic isolates using the Lancefield Grouping Kit.
Results follow.
Interpretation of Lancefield Grouping for β-Hemolytic Unknowns
Unknown
Observations and Interpretations
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Unknown 2
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Unknown 3
13.
Finally, you test your α-hemolytic unknown, Unknown 1, by Lancefield
Group Typing. The results are provided below.
Observations and Interpretations of Lancefield Typing of Unknown 1
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14.
You may have noticed that you felt you knew the identity of one or more
of the unknowns without doing all of the tests. Given that you work in a clinical
lab dealing with dangerous pathogens, why do you think you did all the tests that
you performed in this analytical exercise?
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15.
Based on the analysis you have done, provide your best guess as to the
identity of each of your unknowns in the table that follows.
Final Identification of Unknowns
Unknown
Likely Identity of the Unknown
1
2
3
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When you complete this exercise, save it using the following format:
[First initial and Last name]_MicroLab_Strep Pathogens
For example: JDoe_MicroLab_Strep Pathogens