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Emily Hostetler
Organism isolated from my hair
Staphylococcus epidermidis
Organism isolated from 3rd floor railing
Micrococcus sedentarius
Identifying Environmental Isolates
Introduction
Billions of bacteria are growing everywhere in the environment. Some microbes are
present on humans, in buildings and even in the air we breathe. Microbiologists around the world
work to identify the multitudes of bacteria present in the environment, yet they have only
isolated and researched about 1% of the microorganism population on Earth. As students, we are
able to explore our surroundings, collect bacterial samples, and discover the steps needed to
classify microorganisms. Student involvement is crucial for sharing knowledge and research
about bacteria. Not only is it possible that a pupil will find an unknown organism, but she will
tell her friends and colleagues about the research being performed, and how important bacteria is
for the survival of the world. In my exploration, I isolated colonies from a strand of my hair and
from the stair railing on the 3rd floor of the science center in order to study environmental
bacteria, and bacteria found on the human body. I hypothesize that the organism isolated from
my hair is of the genus Staphylococcus and the species epidermidis, while the microbe isolated
from the railing is of the genus Micrococcus and the species sedentarius because of their
microbial metabolisms, ability to ferment certain carbohydrates, and general growth under
certain conditions.
Materials and Methods
Before the tests can begin, the collection and isolation of particular microbial populations
has to occur. Firstly, a strand of hair was pulled from my head and the 3rd floor railing was
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swabbed thoroughly for bacteria. Then, the microbes from the railing swab and the strand of hair
were aseptically inoculated on separate nutrient agar plates. After incubating at 37°C for 48
hours, the bacterial growth was observed. A colony from the railing bacteria, and a colony from
the bacteria growing around the hair strand, were picked up from the plates and added to a
nutrient broth tube which was incubated at 37°C. The isolates were then streaked for isolation,
and, after incubation once again at 37°C, a yellow colony from the railing bacteria (Isolate Y),
and a white colony from the hair strand (Isolate H), were chosen to be subcultured on slants, and
used for testing throughout the rest of the experiment.
Ultimately, to identify the bacteria, many tests had to be performed to ensure that the
most knowledge possible about the isolates was obtained. One of the most important tests was
the gram stain, because it differentiates the bacteria into either positive or negative, which makes
identification much simpler. Learning about the isolates’ metabolism, or what molecules they use
as an energy and nutrient source, was a significant part of the identification process. Table 1.
demonstrates all of the microbial metabolism tests performed on isolate H and isolate Y. Another
set of essential tests that were performed on the isolates were the carbohydrate fermentation
tests, which can be reviewed in Table 2. The isolates were also tested for their ability to
withstand certain temperatures and osmotic pressures in their environment. These particular
growth conditions may be observed in Table 3. Finally, antibiotic tests were performed on the
isolates using the Kirby-Bauer method to acquire results about what the bacteria may or may not
be able to withstand. The specific antibiotics used in this series of tests are located in Table 4.
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Results
Table 1: Microbial Metabolism
This table represents the isolate's ability to perform certain functions and
processes within the metabolism. "+" indicates a positive test, "-" indicates a
negative test, and a blank indicates that no test was performed.
H
Y
Gelatin Hydrolysis
Urea Hydrolysis
Casein Hydrolysis
Lipid Hydrolysis
Oxidase
Catalase
+
+
Decarboxylase
Arginine
+
Lysine
Ornthanine
Production of H2S (TSI)
Litmus Milk
IMVIC
Indole
no color for either
Methyl Red
+
Simmons Citrate
+
Voges-Proskauer
Coagulase
Nitrate Reduction
+
+
after Zinc added
Starch Hydrolysis
DNase
+
Table 2: Carbohydrate Fermentation
This table indicates whether the isolate was able to ferment certain types of carbohydrates. "No" means that no
growth or acid formed, "acid" means that acid was present, "gas" means that gas formed, and "yes" means that
growth occurred.
Carbohydrate Fermentation for Isolate H
Carbohydrate Fermentation for Isolate Y
Carbohydrates
Acid/Gas
Growth
Carbohydrates
Acid/Gas
Growth
Adonitol
no
yes
Adonitol
no
yes
Arabinose
no
yes
Arabinose
no
yes
Dulcitol
no
yes
Dulcitol
no
yes
Fructose
acid
yes
Fructose
no
yes
Galactose
no
yes
Galactose
no
yes
Glucose
no
yes
Glucose
no
yes
Inulin
acid
yes
Inulin
no
yes
Inositol
acid
no
Inositol
no
yes
Lactose
acid
no
Lactose
no
yes
Mannose
no
no
Mannose
no
yes
Maltose
acid
yes
Maltose
acid
yes
mannitol
no
no
mannitol
no
yes
Raffinose
no
no
Raffinose
no
yes
Rhamnose
no
no
Rhamnose
no
no
Sacchrose
acid
yes
Sacchrose
no
yes
Sorbitol
no
yes
Sorbitol
no
yes
Xylose
no
no
Xylose
no
no
Sucrose
acid
yes
Sucrose
acid
yes
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Table 3: Growth Conditions
This table represents each isolate's ability to grow in
different temperatures and osmotic pressures. "+" indicates
little growth, "++" indicates good growth, and "+++" indicates
much growth, while "-" indicates no growth.
Temperature
4°C
20°C
28°C
37° C
45°C
55°C
H
+
+
+
-
Y
++
++
-
Osmotic Pressure
2%
4%
6%
8%
10%
H
+
+
++
++
+++
Y
+++
++
-
Table 4: Antibiotic Tests
This table indicates the resistance "R," susceptibility "S," and intermediate "I," effects the given antibiotics
have on the isolates. The zone of inhibition was measured from the antibiotic disk to the edge of the bacterial
growth.
Antibiotic Tests for Isolate H
Antibiotic Tests for Isolate Y
Antibiotic
Zone of Inhibition R/I/S
Antibiotic
Zone of Inhibition
R/I/S
Ampicillin
6mm
R
Ampicillin
35mm
S
Bacitracin
30mm
S
Bacitracin
30mm
S
Chloramphenicol
30mm
S
Chloramphenicol
30mm
S
Clindamycin
6mm
R
Clindamycin
6mm
R
Erythromycin
6mm
R
Erythromycin
30mm
S
Kanamycin
30mm
S
Kanamycin
15mm
I
Neomycin
30mm
S
Neomycin
26mm
S
Novobiocin
30mm
S
Novobiocin
30mm
S
Penicillin
6mm
R
Penicillin
6mm
R
Streptomycin
30mm
S
Streptomycin
26mm
S
Tetracycline
30mm
S
Tetracycline
30mm
S
Vancomycin
30mm
S
Vancomycin
30mm
S
Table 5: General Bacteria Morphology
This table includes the general formation and information about
the indicated isolate.
General Bacteria Morphology for Isolate H
color
white
colony morphology cocci
big staph not motile
cell morphology
cocci
Gram
positive
General Bacteria Morphology for Isolate Y
color
yellow
colony morphology cocci
staph
not motile
cell morphology
cocci
Gram
positive
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According to the results, the different isolates do share some similarities, but the
differences in their fermentative and metabolic processes and growth conditions make the
bacteria much unlike each other. For example, when looking at Table 3., one may notice that
Isolate H has much growth in the 10% NaCl suspension and a very low amount of growth in the
2% NaCl suspension, while Isolate Y has much growth in 2% NaCl and good growth in 4%
NaCl, but no growth in any of the other conditions. The isolates demonstrate nearly complete
opposite growing conditions. However, when tested for their optimum growth temperature,
Isolate H and Isolate Y both showed the most growth in the 28°C to 37°C range, with Isolate H
showing some growth in 20°C as well. Therefore, although the bacteria have unique osmotic
pressure needs, their temperature needs are relatively the same. Upon reviewing the carbohydrate
fermentation activity of the isolates in Table 2., it is apparent that Isolate H was able to ferment
and use many more of the carbohydrates than Isolate Y was able to. Isolate Y only fermented
maltose and sucrose, while Isolate H fermented fructose, inositol, lactose, mannose, maltose,
sacchrose, and sorbitol. Table 4., illustrates the isolates’ resistance or susceptibility to a variety
of antibiotics. The results proved to be moderately similar in that Isolate H and Isolate Y were
both resistant to penicillin and clindamycin. Isolate H, however, was also resistant to ampicillin
and erythromycin, and Isolate Y was intermediately affected by kanamycin. The microbial
metabolisms of Isolate H and Isolate Y only have a few dissimilarities shown in Table 1. Isolate
H had a positive methyl red test, a negative Simmons citrate test, and a negative DNase test,
while Isolate Y had the opposite of those tests. Both isolates did test positive for catalase, though
and negative for other important processes such as gelatin hydrolysis, lipid hydrolysis and
oxidase.
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Discussion
The isolated bacteria from the 3rd floor railing, Isolate Y, may be classified as
Micrococcus sedentarius because of the data collected from the tests performed throughout the
experiment. These bacteria are buttercup yellow and found living on the human skin. However,
not all of the data matches exactly to the information gathered from the original species, because
my Isolate Y may have evolved, or may be a slightly different species. The bacteria found in the
genus Micrococcus, are usually gram positive, cocci cells in a staph formation, and are not
motile. Isolate Y fits this description completely, and can also grow in an environment of 5%
NaCl, is catalase positive, and is negative for glucose fermentation, all of which are all basic
identifiers for Micrococcus bacteria. Classifying what species Isolate Y is proved to be slightly
more difficult. I put the most emphasis on the catalase, oxidase and glucose tests, while also
considering in what growth conditions the organism is most likely to grow. Micrococcus
sedentarius was the best match for Isolate Y for a variety of reasons. The starch hydrolysis test,
urease test, nitrate test, and most of the carbohydrate fermentation tests all matched with my
organism. The hydrolysis tests and carbohydrate fermentation tests were important to match
because they demonstrate how the bacteria obtain nutrients in their environment. Of course, not
all of the tests completely agree, which could be because Isolate Y evolved slightly from the
original species. Unlike the original species sedentarius, Isolate Y did not break down gelatin, or
have a positive Simmons citrate test. However, the environments where the bacteria thrive
match the original sedentarius species and prove that Isolate Y is Micrococcus sedentarius.
Research is never complete though. There are multiple tests that could still be performed that
will solidify the evidence for the bacteria. More carbohydrate tests could be performed and the
bacteria could be tested for nitrogen use.
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The isolated bacteria from my strand of hair, Isolate H, can be identified as
Staphylococcus epidermidis. The bacteria are grey/white and are found on human skin and skin
glands. Similar to Isolate Y, there is evidence that Isolate H is Staphylococcus epidermidis but,
because of cell evolution, and the possibility of having a slightly different strand of bacteria, not
all of the tests agreed completely. Staphylococcus bacteria are gram positive, cocci cells, in a
staph, or clustered formation. They are positive for catalase, a very important process in the
bacteria, and like to grow in high levels of NaCl, possibly because of the salty quality of the skin.
The bacteria will also grow in temperatures ranging from 15°C to 45°C which is the general
range for Isolate H. Staphylococcus epidermidis proves to be the best choice for Isolate H
because nearly all of the carbohydrate fermentation tests and microbial metabolism tests agreed
with the information collected from the original species. Isolate H was also susceptible to the
antibiotics neomycin and novobiocin which shows that the species has not changed enough to be
resistant to certain antibiotics. Other tests that can be performed to further prove that Isolate H is
in fact Staphylococcus epidermidis are nitrogen tests, if the cell has lactic acid or even if it uses
acetoin. All of which are defining factors for the cell.
With the billions of bacteria in the world, it is impossible to stay away from microbe sin
everyday life. It is important to learn about the bacteria instead of fearing the strains that are
harmless, and possibly even helpful. Staphylococcus epidermidis and Micrococcus sedentarius
are only two microbes present in the environment. With student and professional research,
hopefully the knowledge of microorganisms in the world will surpass the 1% of known microbes
known today.
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