11/11/2017 Microbiology Unknown Lab Report | E.

coli | Unknown Report

Lab Report | E. coli | Microbiology

Unknown Report
By jay snaric at January 4, 2013 | 7:32 am | Print
Microbiology Unknown Lab Report
DEANA ISSAWI
MICROBIOLOGY

INTRODUCTION
Bacteria lies everywhere and affects multiple people therefore knowing the causes of the bacteria
can help treat an ill patient. In microbiology laboratory class many techniques were studied to learn
how a microorganism will react in certain conditions. The techniques used were applied to identify
an unknown bacterium.

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MATERIALS AND METHODS

The lab instructor gave out an unknown labeled 124, which contained a gram positive bacterium
and a gram negative bacterium. The techniques that were studied in the laboratory were applied to
find the unknown. Throughout the study the course laboratory manual was periodically referenced
to perform the techniques properly.
The first technique that was performed was isolating the unknown broth on an nutrient agar plate,
by streaking the unknown broth using the isolation streak method that was described in the lab
manual. This is needed to be done to separate the two bacteria and to develop a pure culture of
each. The plate was placed in the incubator and the bacteria began to grow. After the bacteria had
completely grown and no isolated pure culture was present another isolation streak was needed to
be performed with the newly grown bacteria. The newly streaked plate was placed in the incubator.
When the bacteria grew and developed an isolated pure culture a gram stain was performed.
Following each step three times in the lab manual on how to perform a gram stain, the bacteria
turned out to be gram negative rod. Since the unknown bacteria was a gram negative rod, a gelatin
test was performed and the organism was inoculated into a gelatin agar tube and placed into the
incubator. If protein was broken down by gelatinase the tube appeared to have white substances in
the tube. The gelatin test with the unknown bacteria had a negative reaction meaning gelatinase
was not produced, which eliminated the bacteria Pseudomonas aeruginosa.
Since the gelatin test was negative a lactose test was performed to detect the carbohydrate
fermentation which leads to acid production. The gram negative bacteria was inoculated into a
lactose fermentation tube and placed into the incubator. The broth starts out as red but if acid is
produced the broth turns yellow. The gram negative bacteria turned the broth yellow meaning acid
was produced, which eliminated the bacteria Proteus vulgaris. Next a Simmons citrate test was
performed to determine if the organism can use citrate as its sole carbon source. The gram
negative bacteria was streaked on the surface of the Simmons citrate agar and was incubated. The
agar starts out to be green but if citrate is used as its sole carbon source then the agar turns blue.
However the inoculated Simmons citrate agar did not turn blue it remained green meaning a
negative reaction occurred.
The negative reaction eliminates the bacteria Klebsiella pneumoniae and Enterobacter aerogenes
for the unknown bacteria and only bacteria left is Escherichia coli. To verify Escherichia coli is the
gram negative bacteria a nitrate test was performed. The bacteria was inoculated into a nitrate
broth and was incubated. The nitrate test determines if nitrate reduces to nitrite. After the broth is
incubated reagent A and B are added to determine if the reduction occurred; if the broth turns red
a positive reaction occurs if not zinc is added. Zinc reduces any nitrate that did not reduce and

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11/11/2017 Microbiology Unknown Lab Report | E. coli | Unknown Report

turns the broth red meaning nitrate did not reduce to nitrite, negative reaction. The unknown gram
negative bacteria had a negative reaction verifying that the unknown bacteria is Escherichia coli.
The unknown broth contains a gram negative bacterium and a gram positive bacterium. The gram
positive bacterium did not isolate on the first nutrient agar plate so in order for the gram positive to
grow it most be placed on a selective medium. The unknown broth was streaked unto a mannitol
salt agar. The bacteria was incubated and the gram positive bacteria began to grow. An isolation
streak was performed on a nutrient agar with the gram positive bacteria and was incubated.
Isolated pure cultures grew and a gram stain was performed. The bacteria appeared to be a gram
positive cocci. A Simmons citrate test was performed it see if citrate was used a sole carbon source.
The Simmons citrate agar was inoculated and incubated, but the Simmons citrate agar did not turn
blue meaning a negative reaction occurred, eliminating Bacillus cereus and Bacillus subtilis. A
nitrate test was performed next to see if nitrate reduces to nitrite. The nitrite broth was inoculated
and incubated which lead to a positive reaction, eliminating Enterococcus faecalis.
Next an urea test was performed. The urea broth was inoculated and incubated. The urea test
detects for the production of urease. The broth initially is yellow but if urease hydrolyzes urea then
the broth turns pink for a positive reaction. The urea broth turned pink meaning urease broke down
urea, positive reaction. A positive urea test eliminates Staphylococcus aureus which leaves
Staphylococcus epidermidis. To verify the gram positive bacteria is Staphylococcus epidermidis a
mannitol test was performed. Mannitol test was performed to detect the carbohydrate fermentation
which leads to acid production. The gram positive bacteria was inoculated into a mannitol
fermentation tube and placed into the incubator. The broth starts out as red but if acid is produced
the broth turns yellow.
The gram positive bacteria did not turn yellow meaning acid was not produced, which verifies
Staphylococcus epidermidis is the unknown gram positive bacteria. All of the tests that were
performed by referencing the lab manual.

RESULTS – (flowcharts were removed from here for formatting reasons)

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CONCLUSION
Several test were performed to conclude the gram negative bacteria was Escherichia coli and the
gram positive bacteria was Staphylococcus epidermidis. Performing a gram stain on gram negative
and gram positive showed that gram negative bacteria was a negative rod and that the gram
positive bacteria was a positive cocci. For the gram negative bacteria a gelatin test was inoculated
to detect if protein was broken down by gelatinase, which it was not eliminating the bacteria
Pseudomonas aeruginosa. Then a lactose test was inoculated and detected if carbohydrate was
fermented which leads to acid production. Acid production did occur meaning carbohydrates were
fermented and eliminating Proteus vulgaris. Next a Simmons citrate was inoculated to identify if
citrate is used as a sole carbon source. Citrate was not used as a sole carbon source, which
eliminated Klebsiella pneumoniae and Enterobacter aerogenes and leaves Escherichia coli as the
unknown gram negative bacteria. To verify that Escherichia coli was the gram negative bacteria a
nitrite was performed. The nitrite test determines if nitrate was reduced to nitrite. A nitrate broth
was inoculated and incubated, afterwards reagent A and B were added to the solution and did not
turn red so then zinc was added to the solution and the broth turned red meaning the nitrate did
not reduce to nitrite. Verifying the gram negative bacteria is Escherichia coli.
The gram positive bacteria was a positive cocci to verify it was a cocci a Simmons citrate agar was
inoculated and incubated. The Simmons citrate had a negative reaction verifying the gram positive
bacteria is a cocci, which eliminates Bacillus cereus and Bacillus subtilis. Then a nitrite test was
performed and nitrate reduced to nitrite having a positive reaction and eliminating Enterococcus
faecalis. Next a urea test was inoculated and incubated. The urea test determines if urease was
produced and breaks down urea. The gram positive bacteria did produce urease having a positive
reaction, eliminating Staphylococcus aureus and leaving Staphylococcus epidermidis.To confirm
Staphylococcus epidermidis is the unknown gram positive bacteria a mannitol test was inoculated
and incubated. Mannitol test detects the carbohydrate fermentation which leads to acid production.
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11/11/2017 Microbiology Unknown Lab Report | E. coli | Unknown Report

Acid was not produce which confirms Staphylococcus epidermidis is the unknown gram positive
bacteria.
Staphylococcus epidermidis lives on the skin andis part of the bodies normal flora. It is also an
opportunistic pathogen that thrives in hospitals by taking advantage of a weakened immune
systems, immunocomprised. (1) Staphylococcus epidermidis is a facultative anaerobe, which means
it can live with or with oxygen. The pathogenic cell produces slime, which is secreted from the
teichoic acid in the cell wall, resulting in a biofilm formation. (2) The infections usually start at skin
wounds caused by catheters, and also effects prosthetic joints and large wounds. Staphylococcus
epidermidis is resistant to most antibiotics like methicillin, novobiocic, and benzyl penicillin, but it is
antibiotic sensitive to vancomycin. (3)

REFERENCES
1. http://www.ebi.ac.uk/2can/genomes/bacteria/Staphylococcus_epidermidis.html
2.
http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html
3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC376388/

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