Laboratory Experiment Growth of Normal flora of the Throat, Nose and Skin

Introduction
We have normal flora in many different areas of the body. The quest to document these areas is ongoing with
the Human Microbiome Project. The different areas of the body have different types of normal flora as we would
expect. The specificity of the normal flora depends on environmental factors such as pH, oxygen concentration, amount
of moisture present, and types of secretions associated with each anatomical site. Normal flora can generally be found:
a. Skin predominantly Staphylococcus is found here. The following organisms can also be found: Streptococci
(alpha-hemolytic, nonhemolytic), diphtheroid bacilli, Enterococci, yeasts and fungi. The organisms vary by skin
location.
b. Eye conjunctiva Staphylococci, Streptococci, diphtheroids and Nesseriae
c. Upper respiratory tract Staphylococci, Streptococci (alpha-hemolytic, nonhemolytic and Streptococcus
pneumoniae), Enterococci, diphtheroids, spirochetes, and members of the genera Moraxella (formerly known as
Branhamella), Neisseria and Haemophilus.
d. Mouth and teeth Anaerobic spirochetes and vibrios, fusiform bacteria, staphylococci, and anaerobic levan-
producing and dextran-producing streptococci responsible for dental caries.
e. Intestinal tract In the upper intestine, predominantly lactobacilli and Enterococci. In the lower intestine and
colon, 96%-99% is composed of anaerobes, such as members of the genera Bacteriodes, Lactobacillus, Clostridium,
and Streptococcus, and 1% to 4% is composed of aerobes, including coliforms, Enterococci, and a small number of
Proteus, Pseudomonas, and Candida species.
f. Genitourinary tract Staphylococci, streptococci, lactobacilli, gram-negative enteric bacilli, clostridia, spirochetes,
yeasts, and protozoa such as Trichomonas species.

In this laboratory we will study the resident flora of the throat, nasal passages and skin. Since these areas of the
body are sources of mixed populations, you will perform streak plate isolations as we have learned in our first two
laboratory projects to separate the different populations. The isolated colonies can then be studied morphologically,
biochemically and microscopically to identify the individual genera of these mixed flora.

The media that we will use to identify the flora in these areas are:
Blood agar plate Blood agar is used to determine the hemolytic properties of the Streptococci and Staphylococci.
Hemolytic reactions can be alpha-hemolytic or beta-hemolytic. If there is no hemolysis then it is termed
nonhemolytic.

Blood agar
Mannitol salt agar plate MSA plates are used to isolate the staphylococci. The non-virulent Staphylococcus
epidermidis grows pink on this plate and the virulent Staphylococcus aureus grows yellow on this plate.
 Sabouraud agar plate SAB is used for the growth of yeast and mold. Yeast cells will develop pigmented or non-
pigmented colonies that are elevated, moist, and glistening. Mold colonies will appear as fuzzy, powdery growths
coming from the mycelium of the mold.

Materials:
Blood agar plate 2 tube of sterile broth four sterile swabs
MSA plate 1 Gram stain reagents Hydrogen peroxide
SAB plate 1 Slides Oxidase reagent

Procedure:
1. Obtain the media that you will need. Label each plate with the type of media, your name, location of area to be
inoculated and the date.

2. Obtain a sterile swab. View the image of the correct method for obtaining a nasal sample. Take one of the
blood agar plates and put it next to you. Take the sterile swab out of the
container. Insert the swab into your nose as illustrated in the diagram. Roll the
swab around in the nasal passage. Remove the swab. Streak one third of your
blood agar with the swab, rolling it as you go. Discard the swab in the
contaminated non-sharp container. Using a sterile inoculating loop, streak the
plate for isolation. Invert the plate. Incubate in the 37oC incubator for 24-48
hours.

3. Obtain another sterile swab. View the image below on how to obtain a throat swab. Take one of the blood agar
plates and put it next to you. Take the sterile swab out of the container. Insert the
swab into the back of your throat and swab the palatine arches or the tonsils. DO
NOT touch the tongue. Roll the swab around the area. Remove the swab. Streak
one third of your blood agar with the swab, rolling it as you go. Discard the swab
in the contaminated non-sharp container. Using a sterile inoculating loop, streak
the plate for isolation. Invert the plate. Place the plate in the candle jar and put
the candle jar into the 37oC incubator for 24-48 hours.
4. Obtain two sterile swabs. Take your SAB plate and your MSA plate and put them on the counter next to you.
Have the sterile broth tube in a test tube rack next to you as well. Select an area of your skin that you wish to
culture. It is best not to use an area like your hands which get washed multiple times in a day. Do not use an
area with make-up or lotion applied. Take the sterile swab out of the container and moisten it with the sterile
broth. Swab a circular area of your skin about one inch in diameter, rolling the swab as you go. Remove the
swab from your skin. Inoculate your MSA plate by rolling the swab over approximately one third of the plate.
Discard the swab in the contaminated non-sharp container. Using a sterile inoculating loop, streak the plate for
isolation. Incubate in the 37oC incubator for 24-48 hours. Take the other sterile swab . Take the sterile swab out
of the container and moisten it with the sterile broth. Swab a circular area of your skin about one inch in
diameter, rolling the swab as you go. Remove the swab from your skin. Inoculate your SAB plate by rolling the
swab over approximately one third of the plate. Discard the swab in the contaminated non-sharp container.
Using a sterile inoculating loop, streak the plate for isolation. Incubate in the 25oC incubator for 48 hours.

5. Take the plates out of the incubator and observe them for growth. Record any and all growth on the plates. For
the blood agar plates, record any hemolysis present. For the MSA plate, record any change in color. Document
the growth on the plates.

6. Perform a Gram stain on each different type of bacterial or yeast colony present using standard gram stain
technique. If mold is present, ask for assistance in preparing your slide using the scotch tape method.

7. Observe the slide and record your results.

8. Identify the gram positive cocci to the genus level using the type of lysis, catalase test, oxidase test and bile test
if needed.
 Name ___________________________________________

Laboratory Experiment Growth of Normal flora of the Throat, Nose and Skin
Observations and Results
1. Document your work up on your throat culture.
a. Insert the image of the plate in the area below. For each colony type, give the type of hemolysis
observed.

The white mucoid clusters showed hemolysis around them, but the agar around the yellow-green colonies, there was no
hemolysis.
 b. Document the gram stain for each different colony type grown on your throat culture. Give the correct
shape and gram reaction for each one.

The white mucoid clusters stained as Gram + cocci arranged in clusters, and the yellow-green colonies were Gram +
cocci in clusters.

c. Identify any gram positive cocci to the genus level using the catalase reaction, oxidase test and bile test.

Both colonies reacted positively to the catalase test, bubbling in hydrogen peroxide. This means that both are forms of
Staphylococcus.

2. Document your work up on your nose culture.

a. Insert the image of the plate in the area below. For each colony type, give the type of hemolysis
observed.

The grey colonies do not show hemolysis, where the yellow colonies did cause hemolysis in the agar around them.
 b. Document the gram stain for each different colony type grown on your nose culture. Give the correct
shape and gram reaction for each one.

Both the yellow and white colonies gram stained as Gram + cocci in clusters.

c. Identify any gram positive cocci to the genus level using the catalase reaction, oxidase test and bile test.

Both colonies bubbled during the catalase, which indicates that they are both forms of Staphylococcus.
 3. Document your work up on your skin culture.

a. Insert the image of the MSA plate in the area below. For each colony type, give the color of the MSA
plate that was observed.

Most of the colonies caused the agar around them to turn yellow, whereas a few remained red.
 b. Document the gram stain for each different colony type grown on the MSA plate of your skin culture.
Give the correct shape and gram reaction for each one.

Both colonies stained as Gram + cocci in clusters.

c. Identify any gram positive cocci to the genus level using the catalase reaction, oxidase test and bile test.

Due to the positive reaction to catalase, both types of colonies are determined to be staphylococcus.

d. Insert the image of the SAB plate in the area below. For each colony type, give a description of the
colony appearance.

The SAB plate grew nothing, so a picture would just be of bare gel.

e. If there are any yeast cells present, gram stain the yeast and record the shape and arrangement of the
cells. If there is any mold present, stain the mold using the method described by your instructor.
Analysis:
1. Did your throat show the typical normal flora? Were there any colonies that were not typical normal flora? List
the typical and atypical normal flora for your throat.

While there was a mixture of many bacteria that appeared in limited numbers, the colonies of my flora that were grown
had one major anomaly, which was a massive amount of Staphylococcus aureus, which is the pathogenic form of Staph
that causes MRSA.

2. Why did we incubate the throat culture in a CO2 jar?

The bacteria that grow in human throats tend to be anaerobes.

3. Did your nose culture demonstrate typical normal flora? Atypical flora? List the typical and atypical normal flora
for your nose.

My nose cultures had only two major colonies of S. Aureus and S. Epidermis, and the presence of S. Aureus

4. Did your skin culture demonstrate typical normal flora? Atypical flora? List the typical and atypical normal flora
for your skin.

For the skin culture, I expected there to be some fungi or yeast, but there was only Staphylococcus, both epidermis and
aureus.

5. Why did we incubate the SAB plate in a 25oC incubator?

Because that is the normal temperature of human skin, which is what the microorganisms prefer to grow in.

6. Did any bacteria grow on the SAB plate? Why or why not?

No, nothing grew on my plate, perhaps because there was not enough on my skin to start a new growth.

7. What value does normal flora have for a host?

It can help preform bodily functions such as digestion, and they also outcompete potentially dangerous organisms.

8. How does the presence of normal flora influence the infectious process?
Some normally benign bacteria that always live inside your body can become virulent if the immune system is
weakened. Most of the time, however, the human flora impedes new infections by outcompeting new pathogens that
are attempting to colonize the body.

Case Studies:

CASE A: Nancy
Nancy is a 6-year-old female. She was taken to her pediatrician for a checkup. As the doctor reviews the childs
history, her mother reports that the child had a severe sore throat several weeks earlier that resolved without
treatment. Upon examination the pediatrician notes that the child has a systolic heart murmur consistent with mitral
insufficiency and suspects that she has rheumatic fever.
a. How was the earlier pharyngitis related to the subsequent development of rheumatic fever?

The antibodies that the body had produced to fight the infection have begun to attack other parts of the body such as
the heart and joints.

b. Rheumatic fever is diagnosed on clinical and serological findings. What test should be done to diagnose
rheumatic fever?

Blood tests should be run to see if there are elevated levels of Streptococcus antibodies in the body.

c. How are rheumatic fever patients treated?

The normal treatment is antibiotics coupled with anti-inflammatory drugs.

CASE B: Pammy
Pammy is a 35-year-old female. She underwent serious abdominal surgery involving extensive bowel resection.
She was maintained postoperatively on a regimen of intravenous broad-spectrum antibiotics. Three days postoperative
she spiked a fever without a clear source. She complains of vaginal discomfort. Blood cultures reveal the presence of an
ovoid cell that reproduced by budding.
a. Based on this observation, what do you think this organism is?
E. coli.

b. Is it part of the normal flora in humans?

Yes

c. How did the treatment with broad-spectrum antibiotics predispose the patient to infection with this organism?
The broad-spectrum antibiotics killed off most of the bacteria that lived in the patients urinary tract and this gave the
normally benign E. coli a chance to spread there.