Bre’yanna Broughton
Bio-2400-04
Lab section day: Thur
Unknown organism #10
Escherichia coli
Unknown Report
After an unknown organism was presented to me, weeks were spent learning and understanding
the concept behind the various biochemical tests which were performed. The data was recorded
onto a table of information as to the positives and negatives; which could be found summarized
on Table 1. Once the table was complete with all information, my investigation endeavor to
finding out the identity of my unknown organism began.
By using Bergy’s manual, the first clue to figuring out the identity was to rule out all organisms
which were gram positives. However, after doing so I was still left with an extensive list of
different options. I referred back to my test results which told me the unknown organism is rod
shaped. This is important, although it did not narrow my search down very much. The unknown
organism established itself to be a facultative anaerobe, that fact changed the whole direction I
was going in.
Through that specific piece of information I was able to determine my unknown organism as
being part of the Enterobacteriacease family, bringing me so much closer to determining the
identity. After locating a specific section of Bergy’s manual, which listed many viable organisms
under the Enterobacteriacease family; I extended my research by ruling out certain genus’s
through test results, and characteristics which could have in no way been my organism because it
did not match up.
As I got closer to my answer, a few organisms proved themselves to be viable such as
Escherichia coli, Shigella boydii, and all species of Yersinia. From there I found what was
practical and what was not by furthering my research on those geniuses provided by Bergy’s
manual. Not only that but also many online databases of microorganisms which allowed me to
further narrow down my choices and by doing so I found that Escherichia coli has to be my
organisms identity.
I believe my unknown organism is Escherichia Coli; of the Enterobacteriaceae family. After an
extensive amount of research which allowed me to narrow down all of my options, E. coli was
the most practical. It doesn’t present any false negatives nor positives and contains all of the
same correct test results that my unknown organism does as well.
Table 1. Biochemical test results of the unknown compared with E. coli
Test Results found for unknown Characteristic result of
#10 Escherichia coli
Gram stain - -
Cell morphology Rods Rods
Pigment White, light yellow -
Oxygen requirement Facultative anaerobe Facultative anaerobe
Glucose fermentation Acid, no growth -
Lactose fermentation No gas, acid, growth [-]
Tryptophan digestion (indole) + [+]
Methyl-red + +
Voges-proskauer - -
Citrate utilization - -
Motility - -
Catalase + +
Cysteine digestion + a
Lysine decarboxylase - b
Gelatin hydrolysis - -
Urea digestion + -
Mannitol fermentation a +
+
tested positive.
-
tested negative.
a
results unknown.
b
26-75% positive.
While there were many tests which helped to prove the identity of my unknown organism, some
tests proved themselves to be more critical than others. All tests were helpful, but some weren’t
as good as the critical ones. The tests which were most crucial were the Gram Stain, Indole
production, Methyl red, Vosges-Proskauer, Citrate, Oxygen requirements, and Catalase
production.
These tests were the basis of my research conducted. Anything which proved itself to be gram
positive, outside of the rod cell morphology family, and not specifically a facultative anaerobe,
were automatically ruled out; Aerococcus, Coprococcus, Deinobacter, and many more, just to
name a few. This left many geniuses such as Alcaligens, Pseudomonas, and Enterobacteriaceae
to be viable candidates. Now, in order to figure out which one my organism was among the
viable candidates, in depth research was done.
The Gram Stain test set the tone and was one of the most important tests. This test showed more
than just the bacteria staining pink or purple; “it showed the chemical and physical properties
of the bacteria’s cell wall (Gram stain, 2021)”. The test gave some insight about the capsules of
the bacteria. It also let me know that since my unknown organism was stained pink, that the
peptidoglycan layer was thin rather than thick; meaning the bacteria did not retain the crystal
violet stain. With it also being stained, the shape was able to aluminate, showing the morphology
of the organism. This further proves or disproves the identity of the organism and allows it to
be identified as one thing and not another. There is a big difference in whether the bacteria is
shaped like beads referred to as cocci, spirals, or rods. Not to mention, if they are singular, come
in clusters, or pairs. This is important information in order to be able to distinguish the different
organisms from one another.
The next test which was just as equally important was the Oxygen requirements test. Through
this test I was able to drastically narrow down my options to figuring out which one my
unknown organism was. As stated before, this test allowed my unknown organism to prove itself
as a facultative anaerobe. Facultative anaerobes are capable of growing with or without the
presence of oxygen.
This could be logically linked with the catalase test, which shows, “there is an enzyme that
catalase the release of oxygen from hydrogen peroxide (Aryal et al, 2018)”.
In addition, the Indole production test proved that the bacteria in my organism was able to
decompose tryptophan; an amino acid, into indole. This was critical information when
distinguishing the few different viable options I had left over. “The indole negative bacteria were
Klebsiella, Serratia, and Yersinia (Indole test, 2020)”. Being that my unknown organism tested
positive, again E. coli was the most appropriate option.
Not to mention, the Methyl red test, which is also a ph indicator. When inside of a broth that’s
being utilized; “If the organism uses the mixed acid fermentation pathway and produces stable
acidic end-products, the acids will overcome the buffers in the medium and produce an acidic
environment. When methyl red is added, if acidic end products are present, the methyl red will
stay red (Douglas et al, 2011)”. In this case, my organism tested positive meaning it was able to
produce acid end products from the glucose.
Now onto Voges-Proskauer, or “The VP test, which detects organisms that utilize the butylene
glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has
been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product
is oxidized in the presence of potassium hydroxide to diacetyl. Creatine is also present in the
reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive
result (Douglas et al, 2011)”.
Through this it is evident that the MR and VP tests can be logically linked together. “ Studies
have also shown that, “Originally the paired MR-VP tests were used to distinguish between
members of the family Enterobacteriaceae, but now they are used to characterize other groups of
bacteria including Actinobacteria (Aryal et al, 2018)”.The positive test results further proves the
fact that the identification of my organisms family is appropriate.
Now concerning the citrate test, “it’s a medium used for the differentiation of
Enterobacteriaceae based on the utilization of citrate as the sole source of carbon. “Bacteria that
can grow on this medium produce an enzyme, citrate-permease, capable of converting citrate to
pyruvate. Pyruvate can then enter the organism’s metabolic cycle for the production of
energy. Growth is indicative of utilization of citrate, an intermediate metabolite in the Krebs
cycle. When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia,
which increases alkalinity (Aryal, 2019)”. This is critical because it’s specific in pointing out the
test results for the different types of species within the Enterobacteriacease family.
Relating this back to my unknown organism, This means that since it tested positive it can use
citrate as the only carbon source, and has a higher alkalinity. Which further confirms its identity.
When reviewing the test results it’s evident that Escherichia coli is citrate negative as well as my
unknown organism but “Enterobacter species are citrate positive (Aryal, 2018)”. This meant
Enterobacter was no longer an appropriate candidate.
Now, In regards to the other tests which were done but weren’t considered critical, those include
the Urea hydrolysis, Gelatin hydrolysis, Catalase production, as well as the Motility tests. These
results matched up with what was tested on my unknown organism. Although they were able to
help further prove the identity of my unknown organism, they weren’t evidence based, and
specifically for E. coli. Through some of these tests it is easy to get a false positive or negative,
so they weren’t necessarily ignored they just weren’t highly taken into consideration.
Now onto the identity of my organism, Escherichia coli better known as E. coli, “are a large and
diverse group of bacteria found throughout the environment, foods, and lower intestines of
healthy people and animals. Most strains of E. coli are harmless however, certain kinds can bring
about troubling symptoms such as diarrhea, pneumonia, urinary tract infection, respiratory
illnesses, and so much more (E. Coli(Escherichia Coli), 2021)”. “E. coli is expelled into the
environment through feces (Escherichia Coli, 2021)”, and in more severer cases it can even
cause death.
“After being studied intensively for about 60 years, scientist have found that they are able to
survive as well as grow outside of its host cell for about 3 days, and under favorable conditions it
takes as little as 20 minutes for them to reproduce (Escherichia Coli, 2021)”. One Interesting fact
regarding E. coli is that, “in 1996 the world saw its worst E. coli outbreak ever. “21 people died
after eating contaminated meat supplied by a butchers shop in Wishaw, Scotland, which was
deemed clean however it wasn’t safe (Health | Sheriff Criticises E. Coli Butcher, 1998)”.
Never the less, not everything about E. coli is completely detrimental. “The harmless strains
which were mentioned before, are part of the normal microbiota of the gut but only about 0.1%.
They can also benefit their hosts by producing vitamin k which helps with blood clotting (Mayo
Clinic Staff, 2020)”. Not to mention, a study conducted by the University of Colorado Boulder
was able to “shed some light on how gut bacteria are beneficial to our health. One key role of E.
coli is that it helps cells within our body to absorb iron (Leitch, 2018)”. So, to a certain extent,
it’s good for us.
Works Cited
Aryal, Sagar, et al. “Catalase Test- Principle, Uses, Procedure, Result Interpretation with
Precautions.” Microbiology Info.com, 11 June 2018, microbiologyinfo.com/catalase-test-
principle-uses-procedure-result-interpretation-with-precautions/.
Aryal, Sagar, et al. “Methyl Red (MR) Test- Principle, Procedure and Result
Interpretation.” Microbiology Info.com, 11 June 2018, microbiologyinfo.com/methyl-red-
mr-test-principle-procedure-and-result-interpretation/.
Aryal, Sagar. “Simmons Citrate Agar- Principle, Uses, Preparation & Result
Interpretation.” Microbiology Info.com, 15 Aug. 2019, microbiologyinfo.com/simmons-
citrate-agar-composition-principle-uses-preparation-and-result-interpretation/.
BERGEY'S MANUAL, 9TH ED, by D. H. Bergey and John G. Holt, Williams & Wilkins, 1993.
Douglas, Gary, et al. Welcome to Microbugz - Methyl Red & Vogues-Proskauer Test,
www.austincc.edu/microbugz/mrvp_test.php.
“E. Coli (Escherichia Coli).” Centers for Disease Control and Prevention, Centers for Disease
Control and Prevention, 11 Mar. 2021, www.cdc.gov/ecoli/index.html.
“Escherichia Coli.” Wikipedia, Wikimedia Foundation, 10 Apr. 2021,
en.m.wikipedia.org/wiki/Escherichia_coli.
“Gram Stain.” Wikipedia, Wikimedia Foundation, 22 Mar. 2021,
en.m.wikipedia.org/wiki/Gram_stain.
“Health | Sheriff Criticises E. Coli Butcher.” BBC News, BBC, 19 Aug. 1998,
news.bbc.co.uk/2/hi/health/154107.stm.
“Indole Test.” Wikipedia, Wikimedia Foundation, 18 June 2020,
en.m.wikipedia.org/wiki/Indole_test.
Leitch, Carmen. “How the E. Coli Bacterium Can Benefit Us.” Labroots, Labroots, Inc., 24 Aug.
2018, www.labroots.com/trending/microbiology/12513/e-coli-bacterium-benefit/amp.
Mayo Clinic staff. “E. Coli.” Mayo Clinic, Mayo Foundation for Medical Education and
Research, 10 Oct. 2020, www.mayoclinic.org/diseases-conditions/e-coli/symptoms-
causes/syc-20372058.
