Jennifer Williams

Escherichia coli
BIOL 2325L Section 1B
April 21, 2015

Introduction:
Microorganisms are the most abundant organisms on Earth. They play many vital roles within our
ecosystem such as decomposition, oxygen production and microorganisms cause infectious diseases within humans,
animals and plants. Prokaryotes are a form of microorganisms and are the basis of every food chain on the planet
(Kellenberger, 2001). Microorganisms are usually unicellular and cant be seen with the naked eye. Although they
are very small they possess the proper functions for life and complete genetic information in their DNA.
Microorganisms are so abundant because of the fact that they can be found in any habitat whether it is extremely
cold or extremely hot. Those microorganisms are called extremophiles and they can live in deserts, rocks, ice and
many other areas of extreme temperatures. If there were no microorganisms on Earth, the ecosystem would have
died by now because they help in degradation of the dead materials (Adnan, 2010).
Identifying microorganisms is important because they affect life in many ways. They can be very beneficial
to humans but they can also cause death. Having as much knowledge as possible about them is important because
microorganisms can influence the food chain, production of medicines and health of plants, animals and humans
both positively and negatively. Types of microorganisms are protozoa, viruses, bacteria, archaea, fungi and algae.
They exist in many different shapes and sizes and have several different uses and functions. These characteristics
along with many others are used to classify each microorganism.
Escherichia coli are one of several microorganisms that have a symbiotic relationship with other
organisms. When humans are concerned, they are not able to digest their food without microorganisms. There is
bacterial specie in our intestinal E. coli which helps to digest the food properly (Adnan, 2010). Identifying E. coli is
important because some are pathogenic and can cause illness.

Materials/Methods
Gram Staining Materials and Methods
Microscope slides
Lens paper
Bibulous paper
Crystal violet
Safranin
Compound microscope
1.
2.
3.
4.

95% ethanol
Grams iodine
Inoculating loop
Slide holder
Clothespin
Immersion oil

A bacterial smear was prepared.

The slide was allowed to dry one minute and the smear was heat fixed.
The slide was flooded with crystal violet and stood for one minute then rinsed with water.
The slide was flooded with Grams iodine and stood for one minute then rinsed with water.

5.
6.
7.

The stain was decolorized by adding 95% ethanol and held at a 45 degree angle and immediately rinsed
with water.
The slide was counterstained with safranin for one minute and rinsed with water.
The stain was carefully blotted and viewed under the microscope with the 10x objective lens then the 100x
objective lens with immersion oil.

Lactose Fermentation Testing Materials and Methods (Selective and Differential Media)
Inoculation loop
Phenyl ethyl alcohol agar (PEA)
Mannitol slat agar (MSA)
Bacti-incinerator
1.
2.
3.

A loop was inoculated with the unknown.

Each agar plate was streaked with the unknown.
The plates were incubated to initiate growth of microorganism.

Citrate Testing
Inoculating loop
Bacti-incinerator
Citrate agar slants
1.
2.

A sterile loop was used to lightly streak a citrate slant with the unknown.
The slant was incubated at 37C for 24-48 hours.

Freeze Thaw Cell Lysis

Dry ice-ethanol bath
Wet ice bath
Thermogloves
95% ethanol in glass test tube
Distilled water
Hot water bath
1.
2.
3.
4.
5.
6.
7.

micro centrifuge tubes

micro centrifuge
vortexer
micropipette
micropipette tips

500L of unknown culture broth was pipetted into micro centrifuge tube
The sample was centrifuged for five minutes at maximum speed.
The supernatant was discarded.
The cell pellet at the bottom of the tube was suspended in 500L of sterile distilled water and vortexed to
get a homogenized solution.
The closed tube was placed in the dry ice-ethanol bath for three minutes to freeze then quickly placed in the
hot water bath for three minutes. This processed was repeated twice for a total of three times.
The sample tube was then centrifuged for five minutes at maximum speed and the supernatant was saved
because it contains the DNA and the pellet was discarded.
The supernatant was transferred to a new sterile micro centrifuge tube and placed in a wet ice bath.

Results/Charts
TEST
GRAM STAIN

OBSERVATIONS
Stained pink with bacilli morphology

RESULTS
Gram negative rods

SELECTIVE MEDIA (PEA)

No visible growth

LACTOSE
FERMENTATION(MSA)
CITRATE UTILIZATION

No visible growth
Color change to green

No growth because unknown is

gram negative
No growth because MSA is used
for mannitol fermentation/gram +
Positive results

FREEZE THAW CELL LYSIS

(BLAST)

All alignments were >=200 (red) and

all possibilities had E values of 0.0

All of the sequencing descriptions

were Escherichia coli

The initial test was a gram stain of the unknown. The first gram stain had inconclusive results because the
stain may have been heat fixed too long and too much of the microorganism was placed on the slide. Another gram
stain was performed and the results were a stain that was pinkish in color and bacillus morphology when viewed
under the microscope. The next test done was with selective media and lactose fermentation. The selective media
used was Phenyl ethyl alcohol which is selective for gram positive bacteria. There was no growth on the media
because the unknown was gram negative. The lactose fermentation was completed with Mannitol salt agar. This agar
was differential for mannitol fermentation and selective for halotolerant gram positive bacteria. The unknown was
gram negative so there was no growth on the agar. The last test completed was citrate utilization. The citrate slant
initially begins blue in color and if it turned green the unknown is positive for citrate utilization. The unknown
citrate testing changed to the color green so the results were positive for citrate utilization. The results of the
unknown testing verified that the unknown was gram negative, fermented for lactose and positive for citrate
utilization. The use of the flowchart and the testing narrowed the choices down the Escherichia coli. The freeze thaw
cell lysis and BLAST confirmed that the unknown was indeed Escherichia coli.

Discussion:
Escherichia coli are a type of bacteria named after Dr. Theodor Escherich. He was a German bacteriologist
who discovered E. coli in the human colon in 1885. This bacterium is referred to as the most studied free-living
organism and the most prevalent in the family of gram negative bacteria (Clark, n.d.). Most E. coli are beneficial and
do not cause harm to humans. On the contrary there are some types of E. coli bacteria, such as Shiga-toxin
producing E. coli that cause some infections other than gastrointestinal infections such as urinary tract infections.
Some types of Shiga-toxin producing E. coli frequently cause severe disease including bloody diarrhea and
hemolytic uremic syndrome which is a type of kidney failure. There are several types of Shiga-toxin producing
E. coli and specifically when outbreaks occurred they were speaking of Shiga-toxin E. coli O157. According to the
CDC the last outbreak in the U.S. was a multistate outbreak in raw clover sprouts in 2014. There have been other
U.S. outbreaks in the past in foods such as ground beef, ready to eat salads and frozen food products (CDC, 2015).

(Leboffe, 2013)

(Owen, 2013)
E. coli is not only naturally in the intestines of humans but the skin also secretes psoriasin, an antimicrobial protein
that kills E. coli. Fingertips of a healthy human can be inoculated with E. coli and within 30 minutes the E. coli is
dead (Owen, 2013).

Conclusion:
Identification of E. coli bacteria or any microorganism was possible throughout the project. The proper
materials were used and tests were done to identify the unknown microorganism. With the proper information about
morphology, staining, and other test results, the identification of the microorganism can be completed. There were a
few minor problems such as over heat fixing a large amount of the initial stained microorganism. The types of
selective media and differential media were poorly chosen because there should have been one choice to show gram
positive negative results rather than two agar types used that are selective for gram positive and mannitol
fermentation rather than lactose fermentation. At one point there was some doubt that the right unknown was used.
The second gram staining proved that the unknown was gram negative. Using the flowcharts and data was very
helpful in determining that the unknown was E. coli. Once the citrate utilization test was completed and the positive
results were observed, this proved the unknown was E. coli. There was further verification done with the freeze thaw
cell lysis and the BLAST which confirmed that the unknown was Escherichia coli because DNA does not lie.

There were all significant matches that resulted in Escherichia coli and the unknown and all the E values for the
results were 0.0. The unknown species was determined, based on the results of Sanger sequencing and BLAST
query, to be Escherichia coli O157-H7: str. SS52 (Accession No. CP010304.1,Altschul et al., 1990).

References:
Adnan, A., (2010) Importance of Microorganisms in the Ecosystem.
http://www.biotecharticles.com/Biology-Article/Importance-of-Microorganisms-in-theEcosystem-232.html/
Altschul, S. F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) Basic local alignment
search tool. J. Mol. Biol. 215:403-410>.
Clark, Marler., (2015). E. coli food poisoning: What is E. coli and how does it cause food
poisoning? http://www.about-ecoli.com/
Escherichia coli: General Information. (2015). Center for Disease Control and Prevention.
http://www.cdc.gov/ecoli/general/
Kellenberger, E., (2001) Exploring the unknown: The silent revolution of microbiology.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1083810/.
Leboffe, M., (2013). Microbiology Laboratory Theory and Application. Englewood, CO: Morton
Publishing.
Owen, J., (2013). Immunology 7th edition. New York, NY:WH Freeman and Company. 145.