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Unknown Lab Report #1
Unknown #2
Andres Benavides
1 May 2017
Professor Whitney
Spring 2017
MCBC 2010 (48M)
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Introduction.
Microbiology is the study of microscopic species that cannot be seen with the
bare eye, this is one of the reasons why microscopes are a crucial tool used in this field of study.
The microorganisms that are studied are generally abundant, unicellular living organisms that
result in beneficial outcomes for some animals and pathogenic for others. Some of this bacterium
makes up part of our normal flora, being non-pathogenic for our organism and helping it in many
biochemical pathways and synthesis progression. Although bacteria may seem harmless at times
by easing processes and protecting tissues, however, opportunistic pathogens may be
encountered in our normal flora which can compromise our immune system and can cause
disease and infection.
Humans are threatened by many diverse types of bacteria that may be
virulent and cause disease. It is important to be able to identify these types of bacteria and
establish a suitable treatment plan and prevention strategy against them. The importance
of identifying certain species of bacteria lies on human’s ability to appropriately fight
against these different diseases and yield an suitable antibiotic to destroy and prevent future
death from the same pathogen. Correct identification of bacteria can also be
valuable in the study of crop production, fermentation of biochemical compounds,
environmental treatments, pesticides, antibiotic production and more. Since microbiology’s
inception, scientists have used a system of classifying to be able to identify the bacteria that we
have discovered so far, classifying them based on their physical appearance, chemical properties,
genetic behavior, components, life spam, among others.
The purpose of conducting this experiment is to be able to identify an unknown bacterial
species through the use of methods learned during this semester in class, and those outlined in
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the lab manual by Dr. Thomas F. Reed and by the application of those various techniques and
experiments learned throughout the semesters. The results showed that the bacterium in question
were Escherichia coli. And Bacillus Subtilis.
Methods and Materials.
All the experiments were completed as described in Dr. Thomas F. Reed manual and
As demonstrated in Microbiology laboratory class, using aseptic techniques. We were instructed
to randomly select a numbered test tube, which contained two different unknown types of
bacteria. Test tube number two was selected. The first experiment that was done was the Gram
stain procedure with the goal of observing the morphology of the colonies, shape and
arrangement of the bacterium, determine its behavior to Gram staining and determine whether
the bacterium fall under the bacillus or cocci strains. Following the gram stain test, a four-way
streak on a nutrient agar plate and an EMB agar was done to further narrow down the options.
Unknown two was applied on two petri dishes for each agar, one to be grown at 25℃ for each
agar, and one to be gown at 37℃ for each agar.
Results.
The first test that was conducted on unknown two was a gram stain test. The staining
microbes with various dyes intensifies the contrast and allows for the observation of cell
structures, size, shape and cell wall types to take place. Gram positive bacteria stain purple and
have a thick layer of peptidoglycan in their cell walls as opposed to gram negative bacteria who
stain pink and have a thin cell wall that’s made up of lipopolysaccharides. The dyes end up
staining differently depending on the thickness of the wall. One gram negative (pink) colony and
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one gram positive (purple) colony were observed. After getting a positive gram stain result from
one of the bacteria in my unknown, I continued with a spore formation test which came back
positive, leading me to the conclusion that one of the bacteria was Bacillus Subtilis.
In the nutrient Agar dishes, colonies of spores formed and observed for dishes at both
temperatures, however neither turned red which allowed me to rule out Serratia marcescens and
continue with and EMB Agar plate to establish further conclusions. After the cultures on the
EMB dishes were given 48 hours to incubate in the two different temperatures, one had a
purplish colony and the other had a purplish colony with a green sheen on it indicating
Escherichia coli.
Test
Gram Stain
Purpose
To determine the
Gram reaction of
the bacterium
Reagents
Crystal violet,
Iodine, Alcohol,
Safranin
Observations
Short Pink rods,
long Purple rods
Spore Formation
To determine if
a sample has
endospores or
not
Malachite
Green, Safranin
Counterstain
Spore formation
observed
throughout petri
dish
Nutrient Agar
To determine the Nutrient Agar,
presence of
Unknown 2
certain bacteria
on the plate.
EMB Agar
To determine the EMB Agar
presence of
Unknown 2
certain bacteria
on the plate
Neither colony
was red, no
reaction was
observed on the
NA plates.
Both petri dishes
yielded
reactions,
however only
one of them had
a green sheen to
it.
Results
One of the bacteria was
observed to be gram
positive and one was
observed to be gram
negative
Spore forming bacteria
was observed leading
to the conclusion of
Bacillus Subtilis
presence in the
bacteria.
Serratia marcescens
not present
Green sheen observed
on petri dish
determining the
presence of
Escherichia coli. In
the unknown sample.
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Unknown 2
Gram stain
Gram Positive
Staphylococcus aureus
Mycobacterium smegmatis
Bacillus subtilis
Spore Test
(positive)
Bacillus Subtilis
Gram Negative
Escherichia coli.
Pseudomonas aeruginosa
Serratia marcescens
Micrococcus luteus
Proteus vulgarus
Culture on NA plate
(Negative)
Culture on EMB plate
(green sheen observed)
Escherichia coli.
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Discussion/Conclusion:
Based on the different sets of tests that were performed and the results that were observed,
it was concluded that the sample concluded Escherichia coli. and Bacillus subtilis. The first test
conducted (Gram stain) yielded results in both the negative and positive bacteria categories. The
gram stain test revealed rod shaped bacillus cells on the sample dish that was being observed under
the microscope. This immediately narrowed down my options to just two possibilities,
Mycobacterium smegmatis and Bacillus subtilis. Following the gram stain positive result, I
conducted a spore formation test whose results were positive for endospores which left only one
possible bacterium, Bacillus subtilis. Now that one of the unknown bacteria had been identified,
the experiment progressed to discovering which unknown bacteria was left inside of the unknown
sample.
After yielding a gram-negative stain for one of the bacteria in the unknown #2 sample,
further testing was needed to draw conclusions on the remaining bacteria. Two nutrient agar plates
were made using a four-way streak method and one was placed in a 25℃-incubation chamber, the
other placed inside of a 37℃-incubation chamber. The respective bacteria were given time to
culture, and after 48 hours it was concluded that neither yielded the red color change on the NA
needed to make Serratia marcescens the remaining unknown. A culture on an EMB plate was
done following the negative NA result and the same procedures were followed. Two plates of
EMB were prepared and placed inside their respective 25℃ and 37℃ incubation chambers. After
giving the bacteria time to culture and produce results, it was observed that the EMB plate that
was incubated in the 37℃ grew a colony of bacteria which had a distinct green sheen to it. Leaving
only one option for my second unknown Escherichia coli.
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After further cross checking of my results with the professor, it turned out that one of my
bacteria was incorrect. Rather than having Bacillus Subtilis, I had Serratia marcescens. There are
several possible explanations for having yielded incorrect results during my experimentation. The
first and always plausible explanation is human error. Even though aseptic technique was used as
described, it is still possible for a foreign bacterium to have been introduced into my unknown
sample bacteria. It is also possible for some of my instrumentation to not have been properly
decontaminated prior to use. I am also not sure of the source of my bacteria or how old they are,
and when trying to distinguish between bacteria, and old sample could stain erratically. A potential
reason for my incorrect gram positive stain is that the smear that I applied to the sample dish was
to thick. This could lead to the sample not being successfully decolorized and make gram-negative
bacteria appear gram-positive or gram-variable.
The Nutrient agar culture test that was done should have yielded a bright red pigment, but
did not, leading to an incorrect result. One of the reasons why this can happen is that if the density
of the bacteria applied to the agar plate is not sufficient, it can reduce the intensity of the pigment
leading to an incorrect observation. Incorrect agar plate making procedures and temperature
fluctuations can be another cause for a faulty test since optimal growth of SA is from 25-27℃.
In the end, the bacteria that were present in unknown test tube #2 were Escherichia coli.
and Serratia marcescens. E. coli bacteria is one of the most studied species and has supplied
scientist with huge amounts of information and has led to many different discoveries and advances.
This bacterium live in the gastrointestinal tract of warm-blooded animals and can be ingested.
Although they live with and inside many different animals and plants and are considered normal
flora, they are also considered to be opportunistic pathogens and can cause harm to humans as
well. Serratia marcescens is classified as an opportunistic pathogen is known more for harm rather
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than good. ubiquitous. It is normally found in soil, water, plants and animals. It is commonly
existing in non-potable water in underdeveloped countries because improper chlorination
processes. This microorganism is a common agent responsible for contamination of Petri plates in
laboratories. While S.marcescens is a pathogenic microorganism, it is generally only harmful o
immunocompromised individuals like those found in hospitals where many of the documented
infections take place. The method of transmission of this microorganism is by either direct contact,
or by catheters, droplets, saline irrigation solutions, and other solutions that are believed to be
sterile.
References:
1. Black, Jacquelyn G., and Laura J. Black. Microbiology: Principles and Explorations. 9th ed.,
Hoboken, NJ, John Wiley & Sons Inc., 2015.
2. Reed, Thomas F., Dr. Microbiology: MCBC-2010 Laboratory Manual. Brevard Community
College, 2010.
3. Newman, Hans. "Microbiology in Pictures." microbiologyinpictures.com, 2015,
www.microbiologyinpictures.com/index.php. Accessed 29 Mar. 2017.
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