Unknown Lab Report
Reagan Grace
Unknown #2
4/23/2024
Group 2 Section 1
Introduction:
In the past few weeks, multiple experiments have been completed to identify an unknown
bacterium that our professor presents us with. Multiple tests have been performed to determine
the cell wall, ability to ferment differing sugars, the ability to move, among other things as well.
A few of the tests that have been performed are gram staining, Eosin Methylene Blue and
MacConkey agar test, glucose and lactose fermentation test, Triple Sugar Iron agar test, Methyl
Red- Vogues Proskauer Test, Sulfide Indole Motility test, citrate utilization test, and urase test.
The Eosin Methylene Blue test, abbreviated EMB, test for the fermentation of lactose
within gram-negative bacteria species. A chemical dye is placed on the agar which prevents
gram-positive bacteria from growing. Bacteria colonies that rapidly ferment lactose turn the agar
plate a green or dark purple color while non-lactose fermenters do not alter the agar’s color.
On the contrary, MacConkey agar is a differential and selective agar test for gramnegative bacteria growth and lactose fermentation. Lactose fermenters change the color of the
agar to a pinkish hue due to the acid produced by fermentation, which in turn alters the pH of the
agar activating the crystal violet within its medium. The lactose fermenter’s color is a bright
pink. Non-lactose fermenters do not change the color of the medium in any way, appearing as
colorless.
Gram staining is a differential stain and is used to determine the difference between
gram-positive and gram-negative bacteria. Gram-negative bacteria appear a pink color due to the
cell wall being broken down and stained by safranin. Gram-positive remains a purple color
because the cell wall does not get broken down and the safranin does not enter the cell.
One way to distinguish between lactose and glucose fermentation is the Durham tube test.
A tube of negative fermentation stays a bright pink color. A positive test for fermentation turns a
yellow color. The Durham tube is a tube inverted at the bottom of the test tube. This tube tests
for gas production during fermentation. If a small bubble of gas gathers at the top of the inverted
tube, then gas has been produced.
The Methyl-Red and Voges Proskauer test, also known in shorthand as the MR-VP test,
are two separate tests. The medium in which is used is the same, but they test for two different
things. The methyl red test tests for glucose fermenters. The medium consists of a special
glucose medium that distinguishes between glucose and non-glucose fermenters. To begin the
test, a species is inoculated within the special medium for a few days. Methyl red is then added
to the medium. If a species can ferment glucose, the medium turns a pinkish-red color. This color
change indicates a lower pH level. If the medium remains a yellow color, then the pH does not
change, therefore indicating that the inoculated bacterium is not a glucose fermenter. The VogesProskauer test checks for the production of 2,3 butylene glycol and acetoin from glucose. The
original medium is inoculated with a bacterium, and then the next step is to add VP reagents. The
tube is then placed on a test tube rack to rest for a short period. A red ring forms at the top of the
tube if the bacterium is positive for the production of glucose. There is no glucose fermentation
present if there is no ring formed at the top of the medium.
The triple sugar iron agar test, shortened to the TSIA test, is a test that assesses for
hydrogen sulfide, fermentation of lactose and glucose, and saccharose. The beginning agar is a
red-colored slant and red-colored butt. The slant is the part of the agar that stems from the butt
and decreases in surface area the further it goes up the tube. The butt of the agar is the part of the
agar that starts at the beginning of the slant and goes to the end of the test tube. The agar consists
of an excess amount of both glucose and lactose sugars. A dye is added to the agar so that when
the pH is raised in the tube the tube turns yellow. This is an indication of either glucose or
lactose fermentation. If the agar changes to black, then hydrogen sulfide is produced by the
consumption of the amino acid cysteine. If after incubation the entire tube remains red, the
bacterium is negative for all that is being tested for. If the butt changes to yellow and the slant
remains red, then glucose is fermented but lactose is not. If the entire tube changes to yellow, the
bacteria ferments both glucose and lactose. The tube also can contain hydrogen sulfide
production, and blackening of agar, while also fermenting glucose and lactose, indicated by the
yellowing of the agar.
The SIM test, also known as the sulfide indole motility test, simultaneously tests for three
different things all in the same medium. Also like the TSIA test, the SIM test determines if a
bacterium breaks down cysteine to produce hydrogen sulfide; also indicated by the black
coloration of the tube. An indole is the byproduct of another amino acid, tryptophan. And lastly,
the ability of the bacteria to move throughout the tube is motility. The SIM medium is inoculated
by stabbing bacteria to the bottom of the agar tube. If the tube remains clear the test is negative
for all three. If the organism produces hydrogen sulfide, the area around the stab will turn black.
Motility can be observed if the tube is cloudy or if growth around the stab has spread throughout
the tube itself. The indole test is the last to be tested. After incubating the tube, reagents are
added, if an indole is produced then the medium will turn pink-red near the surface.
The citrate utilization test examines if a bacteria will survive on citrate as its sole source
of carbon. The medium in the tube is green, if citrate is utilized resulting in a positive reaction,
then the medium will change to a colorful blue.
The urease test is a test based on metabolizing nitrogen. To begin this test bacteria is
inoculated into a urea broth. If the bacteria can produce the enzyme urease, the test is positive.
Positive tests are indicated by the broth color changing to bright pink due to urea being broken
down into ammonia. Negative tests remain orange in color.
The final test that the lab does not get to complete is the oxidase test. This tests if the
bacteria is an aerobic respirator. Positive tests turn the piece of paper purple, while negative tests
do not change the paper’s appearance.
Results:
The results of these multiple experiments are irrefutable. The bacteria is a gram-negative
bacterium. The conclusion of this is the result of gram staining. An isolated colony of the
bacteria is heat-fixed to a slide. Crystal violet dye is added as the primary stain for thirty seconds
and rinsed with water. Iodine acts as the mordant and is then added to the slide for sixty seconds,
and then rinsed off with water. The decolorizer, ethyl alcohol, is added for approximately ten
seconds. After rinsing the alcohol off with water, the last step is to add the counterstain, safranin,
for thirty seconds. Rinse the slide with water once again and examine it under the microscope
once the slide is dry. Upon close inspection through the microscope, it is determined the bacteria
is pink in color, therefore indicating it is gram-negative. The bacteria’s shape looks like a rod
meaning it is a bacillus bacterium.
The second test the bacteria is exposed to is the EMB and MacConkey agar test. This test
is if the bacteria ferment lactose or not. The agar is inoculated with the unknown bacteria. The
plate is placed into an incubator at thirty-seven degrees for forty-eight hours. After incubation it
is evident that the bacteria grew, furthering proof of the bacteria being gram-negative, and
changes to a green color. The green color indicates lactose fermentation. Simultaneously, the
MacConkey agar is inoculated also. The culture grew on the MacConkey agar as well and turned
a bright pink color. These two tests indubitably indicate the bacteria is gram-negative.
The next test is using the Durham fermentation tubes. A flamed loop is used to inoculate
the unknown bacteria into two separate liquid mediums. The first medium is for glucose
fermentation and the other is for lactose fermentation. Tubes are placed into a test tube rack for
one week. Both tubes turn yellow which is an indication of glucose fermentation and lactose
fermentation. The Durham tube also contains a bubble at the top of it. This bubble is gas
production during fermentation.
The MR and VP tests are next on the list. Bacteria are inoculated by a flamed loop. The
inoculated tubes rest for one week. After one week methyl red is added to the test tube. The tube
turned red. The red coloration depicts a positive result for the MR test. The VP test uses the same
procedure as the MR. The VP test should result in a negative because the bacterium can only be
MR or VP positive, but not both. The tube turned red after adding the VP reagents. This is a false
positive. The VP reagents are expired. This causes a false positive. By using reagents that are in
date corrected this mistake. The result for the VP test is negative—no red ring formed at the top
of the tube indicating a positive test.
The triple sugar iron agar test is next to be conducted. This is testing for the bacteria’s
ability to ferment glucose, lactose, sucrose, and hydrogen sulfide production. The bacteria are
inoculated into the test tube by stabbing into the butt as well as streaking the surface of the slant
while also using the same aseptic techniques as before. The tubes rest for one week. The tube
after resting is yellow throughout the agar. The bacteria ferments glucose, lactose, and sucrose.
There is no blackening, so no hydrogen sulfide is produced.
Next is the sulfide indole and motility test. We use the same inoculation technique to
prepare the medium, except for one thing. The SIM test does not have a slant, so there is no
streaking of the slant. The tube is only stabbed to the bottom. This stabbing allows for motility to
be examined. Just as in the TSIA test, there is no black in the tube, therefore there is no hydrogen
sulfide production. The next thing to determine is motility. The bacteria are motile due to the
tube becoming cloudy and spreading away from the stab site. The last thing to check is for
indole. Several drops of Kovac’s reagents are added. The red band that forms at the surface of
the tube indicates a positive indole.
The next to last test is the citrate utilization test. This test is also known as the Simmons
test. This test is to determine if citrate is the lone source of carbon. The same aseptic technique is
used to inoculate the tube. Just like the TSIA the tube is stabbed and streaked. The tubes were
incubated for forty-eight hours. The tube did not change color at the butt, but the color did
change at the slant. This means that the bacterium is positive for citrate.
The last test to be completed is the urease test. Once again inoculate the test tube using an
aseptic technique, incubate it for forty-eight hours, and examine it the following week. Urease is
a liquid medium, so there is no stabbing or streaking to be done. The tube changes to a pink color
if it is a positive test. The bacterium being studied does not have a pink color, instead it remains
orange. This means that a negative result is evident, and the urease is not being broken down by
the bacteria.
The oxidase test is the last test to be completed. We did not do the oxidase test due to a
lack of time left in the class.
Conclusion:
After many tests, the bacteria that is tested for is Escherichia coli. The conclusive results
come from both lactose and glucose fermentation. Only a few bacteria ferment both lactose and
glucose. These bacteria include Enterobacter aerogenes, Escherichia coli, and Citrobacter
freundii. Escherichia coli and Enterobacter aerogenes are negative in hydrogen sulfide
production. The difference between Enterobacter aerogenes and Escherichia coli is the MR-VP,
indole, and citrate tests. Escherichia coli is indole-positive, MR-positive, and citrate-negative.
During our testing, we had a positive test for citrate utilization. This is a false positive.
Escherichia coli is citrate-negative. As a group, we could have allowed contamination in some
way, resulting in this result. Even excluding the citrate test there is zero doubt that the bacteria is
Escherichia coli.
Organismal Report:
Escherichia coli, commonly referred to as E. coli, are facultatively anaerobic, gramnegative, rod-shaped bacteria that naturally live in normally functioning gastrointestinal systems.
Escherichia coli was first discovered by Theodor Escherich in 1885. The optimal temperature of
growth is 37 C. Escherichia coli does not sporulate therefore boiling or any basic sterilization
kills it. There are hundreds of different strains of Escherichia coli. A well-studied strain produces
Shiga-like toxins, which cause severe illness by consuming contaminated meat and cheese. There
are six enteric forms of bacteria, bacteria that live naturally in the intestines. These categories are
based on their virulence properties. These groups are enterotoxigenic E. coli (ETEC),
enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorrageic E. coli
(EHEC), enteroadherent aggregative E. coli (EAggEC), and verotoxigenic E. coli (VTEC). These
enteric bacterial strains cause several different intestinal and extra-intestinal infections such as
urinary tract infection and mastitis. That is not to say that all Escherichia coli bacteria are
harmful. Most Escherichia coli live within our intestines, where they assist in breaking down
food we consume as well as processing waste, vitamin K production, and food absorption. The
harmful part of Escherichia coli is it is the cause of pneumonia. Respiratory tract infections are
uncommon with Escherichia coli due to how close the large intestine is to the lungs. That is not
to say it is impossible. People who suffer from strokes, alcohol disorders, electrolyte
malfunctions, and a few other disorders are at higher risk. Another form of infection by
Escherichia coli is intra-abdominal infections. These types of infections are often the result of
mucosal barrier damage in the gut. This damage leads to local infections such as diverticulitis or
appendicitis. It can also cause distant infections like liver abscesses. Total and complete
disruption of the gastrointestinal tract can be either spontaneous, traumatic, or anastomotic.
Anastomotic is the site where prior surgery has reconnected bowel has failed to heal. Escherichia
coli plays a large role in these types of infections but is not the sole cause unless it has been
isolated from a sterile place.
The genome structure of Escherichia coli is one circular chromosome. Few come with a
circular plasmid also. Lab researchers have completely sequenced its chromosomal DNA. It has
a single chromosome with roughly 4,300 potential coding sequences, and only around 1,800
known proteins. Because of the many different strains, each of the strains differs slightly in its
genotype.
Escherichia coli is easily manipulated and because of this is widely studied. One recent
study is working with the adherent-invasive Escherichia coli (AIEC) which abnormally
colonizes the ileal mucosa of Crohn’s disease patients and invades their epithelial cells. This
study shows that this type of CD-associated AIEC can stick to the brush border of the primary
ileal enterocytes. Another study that has been conducted is the genome evolution. This is a
longer-term study. Genome sequencing done on an experimental population has allowed for a
deeper dive into the relationship between the evolution of the genome and organismal adaptation.
Escherichia coli was grown and sampled for nearly twenty years. The genome was sequenced at
generations 2,000, 5,000, 10,000, 15,000, 20,000, and 40,000 from an asexual population that
evolved with glucose as the limiting nutrient. Comparative genome sequencing showed scientists
there was a difference between the ancestral and evolved genomes that accumulated in an almost
linear fashion, where the fitness trajectory was far from being linear. More specifically, the rate
of fitness improvement decelerated over time. The drift hypothesis unfortunately does not
explain the disagreement in rates of genomic and adaptive evolution. The discrepancy may be
due to clonal interference. Clonal interference is when bacteria generations arise from different
asexually beneficial mutations. The population maintained a low rate of mutation but showed a
slight increase later. This increase is due to the establishment of a mutator lineage. Mutator
lineages are a subset of a population where high rates of mutation occur, this is often the result of
DNA repair and replication. Due to Escherichia coli having such a low mutation rate, there were
no mutations in the first 20,0000 generations. These discoveries are evidence that it is extremely
important to explore the genome evolution and adaptation relationship over long periods.
References:
Lim, J. Y., Yoon, J., & Hovde, C. J. (2010). A brief overview of Escherichia coli O157:H7 and
its plasmid O157. Journal of microbiology and biotechnology, 20(1), 5–14. Accessed 24 April
2024. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645889/
Collier, Ryan P. May 11, 2023. Escherichia coli (E coli) Infections Workup.
Medscape. Accessed 24 April 2024. https://emedicine.medscape.com/article/217485overview?form=fpf
"Escherichia coli." MicrobeWiki, Accessed 24 April 2024.
microbewiki.kenyon.edu/index.php/Escherichia_coli.
Srinivas Garlapati. 2016. Microbiology Laboratory Manual. University of Louisiana at Monroe