Download Indigenous Unknowns Lab Report Introduction

Indigenous Unknowns Lab Report
Introduction:
Normal flora, which make up about 90% of the cells of a human body, are microbes that
live and grow on the body without causing disease under normal conditions. Many of these
bacteria simply live on the body without harming or helping it, but many of the bacteria that
inhabit the body can also protect us against other bacteria that may grow and cause disease. The
purpose of this experiment was to swab the skin, nose, and throat to see what bacteria are present
in each of these bodily environments, and then to choose three colonies and identify these
bacterial species using a dichotomous key and a series of biochemical tests. The identification of
normal flora can be beneficial in the case of an opportunistic infection. If one of the body’s own
normal flora is given the right conditions, such as increased growth or a weakened immune
system, it can cause an infection. In this case if an infection results from one of the body’s own
flora, it may be necessary to identify the organism in order to decide which antibiotic to use.
Each individual person has their own unique combination of microbes living on them as normal
flora, but there are certain genera that are commonplace in certain body locations. Of the places
that were observed in this experiment, the skin and the nose generally have the same genera.
These species include Corynebacterium, Micrococcus, Staphylococcus, and Proprionibacterium.
The bacteria in the throat are generally different from those on the skin because they live in a
different environment under different conditions. These bacteria include Fusobacterium,
Haemophilus, Lactobacillus, Neisseria, Staphylococcus, Veillonella, and the most common
inhabitants of the oropharynx are species of the Streptococcus genus. In this experiment, a
dichotomous key and biochemical tests were used to identify one species from each of the
environments that were swabbed (1).
Methods:
To begin this experiment, five swabs were used to take bacteria from the skin, nose, and
throat. The swab was dipped in sterile water and the back of the hand was swabbed then heavily
inoculated onto the primary area of a blood agar plate. This was then done for the inside of the
nares. To swab the back of the throat, the swab was not dipped in sterile water as the
environment is already moist, and the blood agar plate was inoculated in the primary area. Then
an inoculating loop was used to finish streaking out each of the blood agar plates. After these
plates were allowed to incubate, five colonies were chosen out of all the colonies from the three
plates combines. Two colonies from the skin plate were used, one from the nose plate, and two
from the throat. These five colonies were then narrowed down to the three bacteria to be
identified: two from the skin and one from the throat. The blood agar plates were observed for
hemolysis and gram stains were performed. A catalase test was done on each bacterial species by
placing a colony onto a slide using a sterile stick, and then dropping hydrogen peroxide onto the
colony. All three bacteria were also streaked onto MacConkey and CNA plates using an
inoculating loop (2).
Next, an anaerobic test was done by streaking skin bacteria #1 onto two plates, and one
plate was put right into the incubator while the other plate was put into an anaerobic chamber
that was placed in the incubator. Three CTA carbohydrate tubes were inoculated with the skin
bacteria: sucrose, maltose, and mannitol, and this was done by using the loop to inoculate the
bacteria into the carbohydrate tube. A modified oxidase test was performed by using a stick to
pick up a single colony and place it onto a microdase disk. A nitrate test was performed by
inoculating a nitrate reduction tube. Then after the tube had been incubated for 24 hours, 10
drops of reagents A and B were added to the broth. An esculin tube was also inoculated, as well
as a coagulase tube, by using a loop to inoculate the bacteria into the broths. A urease slant was
also done by streaking the slant with bacteria using a loop. An arginine dihydrolase test was done
by inoculating a Moeller decarboxylase tube as well as an arginine tube with bacteria and putting
a layer of mineral oil in each of the tubes. A mannitol salt agar plate was also used by streaking
down the middle and then crossing over the first streak several times. Finally, a DNAse plate was
done by streaking two parallel lines about an inch apart from each other (2, 3).
For skin bacteria #2, only two additional tests were performed. A modified oxidase test
and a glucose tube were inoculated using the same procedures that were used for the skin
species. Finally, for the throat bacteria, mannitol and lactose tubes were inoculated using the
previously mentioned procedure. An arginine tube was also inoculated as well as an esculin
broth, a nitrate broth, and a urease slant. A DMS deep was also inoculated by taking a needle and
stabbing the bacteria to the bottom of the tube (4).
Results:
First, all three bacteria were streaked onto blood agar, TSA, MacConkey, and CNA
plates. On the TSA, skin bacteria #1 appeared as small, white, round colonies that were about
2mm in size, and there was no hemolysis on the blood agar plate because there was no clearing
around the individual colonies. When streaked on MacConkey and CNA plates, the bacteria
grew on CNA but did not grow on MacConkey. Next, a gram stain was performed on this
bacteria, and the bacterial cells were small cocci that were purple and in clusters, making it a
gram positive coccus. The catalase test was positive, which was indicated by the production of
bubbles after hydrogen peroxide was added to the colony. An anaerobic test was also performed,
and because the bacteria grew in the incubator as well as the anaerobic chamber, which means it
is a facultative anaerobe or aerotolerant and can grow in both anaerobic and aerobic conditions.
The carbohydrate broths that were inoculated with this bacteria all remained red and did not turn
yellow, so skin bacteria #1 was negative for sucrose, maltose, and mannitol. After the bacteria
colony was put onto the microdase disk for the modified oxidase test, it did not turn purple,
indicating a negative result. For the nitrate test, the broth turned a deep red after the addition of
both reagents, so this is a positive nitrate result. The esculin test also yielded a negative result
because after incubation the broth did not turn black and remained its original color. The
organism also does not have urease because the urease slant did not turn bright pink after
incubation. The organism tested negative for coagulase because the liquid in the tube remained a
liquid and did not solidify. The broth in the Moeller decarboxylase tube turned yellow, indicating
that the organism grew in the tube and allowing the arginine test to be interpreted. The organism
was positive for arginine because the broth in the tube was purple after incubation. On the
mannitol salt plate, the organism grew on the plate but was also able to ferment mannitol salt,
indicated by the yellow precipitate around the growth. There was no clearing between the streaks
on the DNAse plate indicating that the organism was negative for the enzyme DNAse. These
results in collaboration with the dichotomous key allowed the identification of this species to be
made as Staphylococcus saccharolyticus (Table 1)(5).
For skin bacteria #2, the appearance on the TSA plate was much different than skin #1.
The colonies appeared large, round, and yellow, and were 4mm in diameter. On the blood agar
plate there was clearing around the growth that did not have a green coloration making it a Beta
hemolysin. The gram stain also showed gram positive cocci that were very small. The catalase
test for this organism was also positive, and it grew on CNA but did not grow on MacConkey.
Finally a glucose test was performed, but because the CTA tube did not turn yellow, the
organism does not ferment glucose and its identity is Micrococcus luteus (5).
Finally, the throat bacteria looked similar to skin bacteria #1 with the small, white
colonies, but these colonies were only 0.5mm in diameter as opposed to 2mm. The blood agar
plate had no clearing around the colonies and therefore had no hemolysis. The gram stain also
showed gram positive cocci but they were in chains. This organism also grew on CNA but not on
MacConkey, and when the catalase test was performed, there were no bubbles indicating that this
organism does not produce catalase. The mannitol test produced a negative result because the
broth did not turn yellow, but the lactose broth was yellow after incubation, indicating a positive
result. The arginine dihydrolase test yielded a negative result because the broth was yellow after
incubation (with a yellow control tube). The esculin broth was a dark black color which is a
positive test result. The nitrate broth did not turn red after adding the reagents so this means the
organism does not convert nitrate to nitrite. The urease tube remained the original color and did
not turn bright pink, so the organism does not contain urease. Finally, there was no pigment in
the DMS tube and the deep stayed the same color, which means the organism did not produce a
pigment. The identity of the organism was determined to be Streptococcus salivarius (Table
3)(5).
Table 1: Skin Bacteria #1
TSA Colony Morphology
Small, White, Round, 2mm
Blood Agar (Hemolysis)
Gram Stain
Catalase
MacConkey
CNA
Anaerobic Test
Sucrose
Maltose
Mannitol
Modified Oxidase
Nitrate
Esculin
Urease
Coagulase
Arginine
Mannitol Salt Agar
DNAse
Identity of Bacteria
Gamma
Gram Positive Cocci, Clusters
+
No Growth
Growth
+
–
–
–
–
+
–
–
–
+
+/Yellow Precipitate
–
Staphylococcus saccharolyticus
Table 2: Skin Bacteria #2
TSA Colony Morphology
Large, Round, Yellow, Raised, 4mm
Blood Agar (Hemolysis)
Beta
Catalase
+
Gram Stain
Small, Gram positive Cocci, Clusters
MacConkey
No Growth
CNA
Growth
Modified Oxidase
+
Glucose
–
Table 3: Throat Bacteria
TSA Colony Morphology
Very Small, Round, White, Smooth, 0.5mm
Blood Agar (Hemolysis)
Gamma
Catalase
Gram Stain
MacConkey
CNA
Mannitol
Lactose
Arginine
Esculin
Urease
Nitrate
DMS
–
Gram Positive Cocci, Chains
No Growth
Growth
–
+
–
+
–
–
–
Skin 1: Staphylococcus saccharolyticus
Skin #2: Micrococcus luteus
Throat: Streptococcus salivarius
Figure 1: Gram Stains
Discussion:
The purpose of this experiment was to swab the skin, nose, and throat for normal flora,
and identify these bacteria using biochemical tests and dichotomous key. The results of the gram
stain indicate that all three bacteria were gram positive cocci. Because the cells of skin bacteria
#1 were arranged in clusters and not chains, this indicates that the genus of this organism may be
Staphylococcus rather than Streptococcus. Because MacConkey supports the grown of gram
negative bacteria, and CNA supports the growth of gram positive bacteria, this organism is gram
positive, confirming the gram stain results, because it grew on CNA and not on MacConkey. The
catalase test is crucial in separating the Micrococcaceae from the Streptococcaceae. The
Micrococcaceae are all catalase positive and the Streptococcaceae are all catalase negative. This
organism must be a Micrococcus or a Staphylococcus because of the positive catalase result.
This organism is likely a Staphylococcus because it has small white colonies, and it also can
grow anaerobically. The dichotomous key for anaerobic Staphylococci was used to identify this
organism. First, the sucrose test was used to narrow down the large group of bacteria to
Staphylococcus cohnii, Staphylococcus carnosus, Staphylococcus saccharolyticus, and
Staphylococcus caprae. The negative maltose result rules out S. cohnii and S. caprae. Finally, to
differentiate between the last two, the mannitol result was used. Because the organism did not
ferment mannitol, its identity was determined to be Staphylococcus saccharolyticus. Because this
organism is a staphylococcus, it is a common inhabitant of the skin and could likely also be
found in the nose. It is normal flora, so it will likely not cause disease unless the immune system
is suppressed but in the case of an opportunistic infection, it could cause diseases such as
pneumonia or endocarditis (6, 7).
Skin bacteria #2 had a similar gram stain to #1, with gram positive cocci arranged in
clusters. The grown on CNA but not MacConkey confirmed that this organism was gram
positive, and there was beta hemolysis on the blood agar plate. The positive catalase test
indicates that this organism is a Micrococcaceae. The yellow colonies suggest that it is a
Micrococcus and based on the dichotomous key this narrows it down to just Micrococcus
varians and Micrococcus luteus. The glucose test was used to differentiate between these two
and because the organism was negative for glucose fermentation, it was identified as
Micrococcus luteus. This result is also confirmed by the positive modified oxidase test. M. luteus
is an extremely common skin and organism that can also be found in dairy products as well as on
dust particles in the air or on surfaces. This organism also generally does not cause disease, but
in the case of an opportunistic infection it may cause an infection wherever it is able to grow in
higher quantities in the case of an immunocompromised patient (8).
Finally, the throat bacteria also appeared as gram positive cocci, but these cells appeared
in chains rather than clusters which suggest that they may be Streptococci. Again, the growth on
CNA but not on MacConkey confirmed a gram positive organism, and it was narrowed down to
Streptococci because it was catalase negative. This narrows it down to the organisms on the
Streptococci dichotomous key. The lack of hemolysis narrows the group of Streptococci down to
Streptococcus rattus, Streptococcus mutans, Streptococcus ferus, Streptococcus cricetus,
Streptococcus sobrinus, Streptococcus salivarius, and Streptococcus milleri. The lack of
mannitol fermentation narrowed this group down to just two organisms: S. salivarius and S.
milleri. Then, the positive lactose result indicated that the identity of this organism is
Streptococcus salivarius. This species is a common oropharyngeal normal flora species that
could cause dental plaque or gum disease if allowed to grow and multiply enough in the absence
of proper hygiene. The identification process of bacteria is not without flaw and error. An error
in inoculation could lead to a false negative if there were no organisms present in the test. A false
positive could also result if too much inoculation occurred. Any errors in reading the tests or
following the dichotomous key would also affect the results and change the ultimate
identification of the organism.
Works Cited
1. Eggers, Christian. Isolation of Indigenous Microorganisms. Hamden: Quinnipiac University,
2011. Print.
2. Eggers, Christian. Micrococcaceae. Hamden: Quinnipiac University, 2011. Print.
3. Eggers, Christian. Gram Positive Rods. Hamden: Quinnipiac University, 2011. Print.
4. Eggers, Christian. Streptococcaceae. Hamden: Quinnipiac University, 2011. Print.
5. Leboffe, M. J., and B. E. Pierce. A Photographic Atlas for the Microbiology
Laboratory.Englewood, CO: Morton, 2005. Print.
6. Wu, Xiaomei, Chunyan Yu, and Xinyan Wang. "A Case of Staphylococcus Saccharolyticus
Pneumonia." International Journal of Infectious Diseases 13.2 (2009): E43-46. Print.
7. Westblom, T. U., Geoffrey J. Gorse, Thomas W. Milligan, and Andrew H. Schindzielorz.
"Anerobic Endocarditis Caused by Staphylococcus Saccharolyticus." Journal of Clinical
Microbiology 28.12 (1990): 2818-819. Print.
8. "Micrococcus Luteus." Wrong Diagnosis. 29 Apr. 2011. Web. 05 May
2011.<http://www.wrongdiagnosis.com/medical/micrococcus_luteus.htm>.