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Lab 17: Bacteria of the Respiratory Tract
Objectives
•
List representative microbiota of the respiratory tract
•
Differentiate streptococci, particularly pathogenic streptococci based on
biochemical testing
Background
The respiratory tract can be divided into two systems: the upper and lower respiratory
tracts. The upper respiratory tract consists of the nose and throat, and the lower
respiratory tract consists of the larynx, trachea, bronchial tubes, and alveoli. The lower
respiratory tract is normally sterile because of the efficient functioning of the ciliary
escalator and the patrolling action of alveolar macrophages. The upper respiratory tract is
in contact with the air we breathe, and it is therefore contaminated with microorganisms.
The throat is a moist, warm environment, allowing the growth of many bacteria. The
normal microbiota of the throat includes genera such as Staphylococcus, Streptococcus,
Neisseria, and Haemophilus. Microbial antagonism keeps the populations “in check,”
suppressing the growth of potentially pathogenic microorganisms.
Streptococci
Streptococci are Gram-positive, catalase-negative bacteria. Many are facultative anaerobes.
Streptococci can be identified by different criteria:
•
hemolytic reactions (their actions on red blood cells)
•
their sensitivity to certain antibiotics
•
other biochemical reactions
•
their surface antigens (Lancefield groups)
Hemolysis
When inoculated on blood agar, streptococci can exhibit three different patterns of
hemolysis:
1. Partial or alpha-hemolysis – (greenish color around the colonies, due to the
oxidation of hemoglobin by hydrogen peroxide produced by the bacteria).
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2. Complete or beta-hemolysis – (clear zone with an edge around the colonies): this is
caused by hemolytic toxins called streptolysins. There are two types of streptolysin:
Streptolysin O (SLO) and streptolysin S (SLS).
3. No hemolysis (gamma) – no change on the agar.
Alpha-hemolytic streptococci include the pneumococci
(especially Streptococcus pneumoniae) and the group of
so-called viridans streptococci (although this name is
falling into disuse). They are usually non-pathogenic
streptococci. S. pneumoniae can be additionally
distinguished by its sensitivity to Optochin, resistance to
Bacitracin, and bile solubility.
Figure 1. Three patterns of hemolysis,
from left to right: alpha (partial), beta
(complete), and gamma (no hemolysis).
(Hemolysis (microbiology), 2022) (CC
BY-SA 3.0)
Beta-hemolytic streptococci are classified further using
the presence of specific cell surface molecules called the
Lancefield antigens. In the 1930s, Rebecca Lancefield
noted that different antibodies were produced
depending on certain proteins present in the cell walls of streptococci. The separation into
Lancefield groups is still a useful method to identify streptococci.
The most important Lancefield groups are A and B, but there are also groups C, D, F, and
G.
Group A Streptococci (GAS) is basically Streptococcus pyogenes, the causative agent of
many pathologies including bacterial pharyngitis (strep throat), impetigo, scarlet fever,
necrotizing fasciitis, and streptococcal toxic shock syndrome (STSS). In contrast to S.
pneumoniae, S.pyogenes is sensitive to Bacitracin.
Group B streptococci (GBS) has as its only member S. agalactiae. It is a human pathogen
affecting mainly neonates and immunocompromised individuals.
Group D streptococci include, among others, members of the Enterococcus family (they
were grouped in their separate genus in 1984). E. faecalis (formerly S. faecalis) and E.
faecium are major causes of nosocomial infections and are of concern because of their
resistance to multiple antibiotics.
Identification of streptococci often takes place using serology and rapid test kits.
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Lab Activity:
Throat Culture
Materials
•
Blood agar plates
•
Toothpicks
•
Sterile cotton swabs
•
Hydrogen peroxide
Procedure
Perform the following steps during the first class period:
1. Start by swabbing your throat. The area to be swabbed
is the back of the throat, between the “golden arches”
as shown in Figure 2. Do not touch the tongue with the
swab.
2. Swab half of the agar plate. Discard the swab in the
biohazard container. Streak the remainder of the plate
using the loop.
3. Incubate the plate, inverted, for 24 hours at 35°C.
Figure 2. Illustration showing
where to swab the throat.
Perform the following steps during the second class period:
1. Observe the plate for hemolysis (hold the plate against the light for better
observation).
Note: Treat the cultures as potential pathogens; follow aseptic technique and discard
everything in the biohazard container!
Lab Activity:
Streptococcus Identification (Supplementary)
Cultures
•
S. pyogenes
•
S. pneumoniae
Materials
•
Blood agar plate
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Disks of optochin and bacitracin
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10% bile salts
•
Gram staining reagents
Procedure
Perform the following steps during the first class period:
1. Draw a line in the middle of the blood agar
plate bottom.
2. Use a swab to inoculate each half with one of
the streptococcus cultures. Do not let the
sides touch. Discard the swab in the
biohazard.
3. Place and press a bacitracin and an optochin
disk on each half using a sterile forceps (see
Figure 3).
4. Incubate the plate for 24 hours at 35°C.
Figure 3. Setup of the demo plate.
Perform the following steps during the second class period:
1. Record your observations regarding hemolytic properties and antibiotic sensitivities.
2. Prepare Gram stains from each organism. Make a diagram/take pictures.
3. Using a sterile loop, prepare a suspension of each organism in a tube of nutrient
broth.
4. Add a few drops of 10% bile salts to each tube. Observe after 15 minutes for lysis of
the cells.
5. Record your observations in the Report table below.
Report
Provide the following
1. Please describe your throat culture in the space below. Include types of hemolysis
observed.
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2. Fill out the following table for S. pyogenes and S. pneumoniae. Use your textbook or
other sources to find the information.
S. pyogenes
S. pneumoniae
Gram, shape, and
arrangement
Hemolysis on blood agar
Sensitivity to optochin
Sensitivity to bacitracin
Bile solubility
Lancefield classification
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References
Hemolysis (microbiology). (2022, April 1). In Wikipedia.
https://en.wikipedia.org/wiki/Hemolysis_(microbiology)
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