Investigation of Unknown Bacterium #02
Investigation of Unknown Bacterium #02
Carla Keeley
BACT: Microbiology
Prof. Malaer
Spring 2014
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Investigation of Unknown Bacterium #02
Introduction
Biochemical tests are an important and necessary factor in discovering the identity of an
unknown bacterium. These tests are performed by inoculation of the species followed by the
analysis of the biochemical reactions of the unknown bacterium (Oliver 91). A Gram stain of the
assigned bacterium is performed which is immediately followed by close observation of the
bacterium under a light microscope. Interpretation of the results will determine whether the
bacterium is Gram negative or Gram positive. Once each biochemical media is inoculated it is
then incubated at 37 degrees for 48 hours where it is subsequently removed from the incubator
allowing interpretation of the biochemical reactions. Upon interpretation, it is determined that
unknown bacterium #02 is identified as Pseudomonas aeruginosa. Infections caused by P.
aeruginosa most often occur in people admitted to hospitals and those with weakened immune
systems. This particular bacterium can also cause infections of the blood, pneumonia, as well as
infections followed by surgery leading to severe illness and in some cases death (CDC 2013).
Materials and Methods
There were a total of seven tests performed to determine the bacterium: 1 Gram stain and
6 biochemical tests. Pertaining to the Gram stain, if the stain appears to be a pink color it is
classified as Gram negative, whereas, if the stain appears to be of a purple color it is classified as
Gram positive. One of the biochemical test performed on the unknown bacterium was
gelatin/protein hydrolysis. Once a sample is taken from the organism, which is done by gently
scrapping the surface of the media with an inoculation needle, it is then placed into the gelatin
deep tube with the pure culture allowing the bacteria to adhere to the media. Once this process is
complete, it is inoculated at 37 degrees for 48 hours. Once incubation is complete, it is placed
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Investigation of Unknown Bacterium #02
into a refrigerator and then removed after 30 minutes. If the media remains liquid after
refrigeration it can then be determined that the bacterium is positive for the presence of
gelatinase. When bacteria that specifically produce the enzyme gelatinase is grown in a gelatin
media, the enzyme hydrolyzes the gelatin molecule and the medium cannot solidify, even at a
cold temperature. Nevertheless, if the media solidifies it indicates that the bacterium is negative
which interprets the absence of the enzyme (Oliver 97).
Another test that was performed on the unknown was methyl red. The media used for
this test is MRVP broth which is used to indicate the acidic level of the unknown organism. If
there is a positive reaction it indicates that the glucose has been metabolized creating the
presence of a highly acidic end result such as acetic or lactic acid, however, if there is a negative
reaction it indicates that the glucose has been broken down by another pathway, leading to a less
acidic compound (Oliver 94). The broth is inoculated with a pure culture where it is later
incubated at 37 degrees for 48 hours. Once incubation is complete, the broth is removed and 5
drops of methyl red reagent is added to the media. A color reaction should happen within the
first 30 seconds of the added reagent. This color change will ultimately determine whether it is
positive (the media turns red) or negative (the media turns yellow or orange.)
Citrate utilization was also one of the biochemical test conducted in this experiment. In
order for the microorganism to utilize citrate it must produce both citrate permease and citrase.
With this test a slant of Simmons citrate agar, which contains the indicator bromthymol blue, is
used for the media where inoculation of the slant occurs by stabbing and streaking the surface of
the agar. Simmons citrate is a synthetic media in which sodium citrate is the only available
carbon source and nitrogen is supplied by ammonium salts instead of amino acids (Oliver 94).
The Simmons citrate slant is then incubated for 48 hours at 37 degrees where it is then removed
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Investigation of Unknown Bacterium #02
and examined for a color change. If the slant remains a green color it indicates that the organism
did not grow therefore resulting in a negative reaction. If the slant turns either partly or
completely blue it indicates that the organism did grow and there was a visible change in the
media resulting in a positive reaction.
Urea hydrolysis was another test performed on the unknown. Urease test is used to
determine the ability of an organism to produce the enzyme urease, which splits ammonia from
the urea molecule and hydrolyzes urea (Acharya 2013). In this test the media used is urea broth
which is created from a solution of yeast extract combined with urea. Once a sample of the
unknown organism is taken, the sample is then loop inoculated and put into an incubator at 37
degrees for 48 hours. Following this process the broth is then examined for a pH color reaction
using phenol red as the indicator. If there is a positive reaction the ammonium that is released
increases the pH level resulting in the broth turning a vibrant, hot pink color. This indicates the
presence of the enzyme urease which is a positive reaction, however, if the broth remains a
yellow to straw color it indicates that there was no enzymatic activity resulting in a negative
reaction (Oliver 98).
Another biochemical test performed was nitrate reduction. This test is used to determine
the ability of an organism to reduce nitrate to nitrites or free nitrogen gas. It involves the enzyme
nitratase. The reduction of nitrate to nitrite and to nitrogen gas takes place under anaerobic
conditions, where an organism utilizes nitrate as its final electron acceptor instead of oxygen
(Oliver 103). This test requires a trypticase nitrate broth to be used for the media. The nitrate
tube is loop inoculated then incubated at 37 degrees for 48 hours where it is then removed from
the incubator and evaluated for results. In order to evaluate the results, the proper reagents are
added first to the broth to determine whether it is a positive nitrate reduction or a negative nitrate
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Investigation of Unknown Bacterium #02
reduction. This process occurs by adding 5 drops of a-naphthylamine, or solution A, and 5 drops
of sulfanilic acid, or Solution B, to the media. Once added, the media must then be shaken
lightly to allow the reagents to react. If the media appears a vibrant, red color upon examination,
then it can be concluded that it is a positive test which means the organism has reduced nitrate to
nitrite. However, if no red color develops and the broth remains colorless then a small amount of
zinc is added to the media. This is done in order to distinguish between the two remaining
possibilities; if the nitrate has remained unaltered (negative reaction), or if it has been reduced to
ammonia of free nitrogen (positive reaction). A negative nitrate reduction will result in the
media turning red, whereas, a positive nitrate reduction results in the media remaining colorless
or turning black (Oliver 104).
Catalase was also one of the biochemical tests conducted in this experiment. This
biochemical test requires a trypticase soy agar plate, also known as a TSA plate, to be used as the
media. First, the unknown bacterium is transferred from the given media to the TSA plate by
forming a single line using an inoculation loop. Once this step is complete, the TSA plate is then
incubated for 48 hours at 37 degrees. The plate is then removed from the incubator and 3-4
drops of 3% hydrogen peroxide, which is toxic to the bacteria’s enzyme system, is placed on the
organism in order to determine whether the enzyme catalase is produced. If the organism
produces bubbles immediately it is catalase positive, however, if it does not produce bubbles it
can then be determined that the test is catalase negative (Oliver 101).
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Investigation of Unknown Bacterium #02
Results
Biochemical Tests/ Gram Stain Results
Biochemical
Positive Results
Negative Results
Tests
Unknown
P. aeruginosa
Bacterium
Results
Gelatin/
Remains liquid
Solidifies after
protein
after refrigeration
refrigeration
Turns red
Turns yellow/
Negative
Negative
Positive
Positive/
hydrolysis
Methyl red
orange
Negative
Turns blue
Remains green
Positive
Positive
Turns light pink/
Remains yellow/
Negative
Positive/
hydrolysis
hot pink
straw color
Negative
Nitrate
Turns red
Remains colorless Positive
Positive
Bubbles
Does not bubble
Positive
Positive
Coccobacillus
Rod (-)
Rod (-)
Citrate
utilization
Urea
reduction
Catalase
Gram Stain
(rod), pink color
Once the tests are complete, the results are evaluated and recorded for further analysis of
the bacterium. One of the tests performed on the unknown organism was a Gram stain. The
results of this test depicted characteristics of a Gram negative component because of the pinkishcolor and rod-like shape; however, it was quite difficult at first glance to determine the actual
shape of this particular organism. According to the text, this particular organism is classified as
a coccobacillus, which means it is a type of bacterium with a shape that is between cocci and
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Investigation of Unknown Bacterium #02
bacilli (Bauman 2009). It is often mistaken for cocci due to its very short rods, which is the
mistake that occurred when the results were originally interpreted. The gelatin/protein
hydrolysis test performed resulted in a negative reaction meaning it did not contain gelatinase.
With this test, the gelatin tube was still in liquid form when removed from the incubator but after
being refrigerated for 30 minutes it solidified. The biochemical test methyl red resulted in a
positive reaction turning the broth a red color. The citrate utilization test resulted in a positive
reaction subsequently turning the slant a blue color. Another test performed on the unknown
organism was urea hydrolysis which resulted in the broth maintaining its initial yellow color
interpreting a negative reaction. A nitrate reduction test was also performed on the organism
which read positive for nitrate reductase creating a red color in the broth. The final determining
factor in identifying this organism relied on the last biochemical test that was conducted, which
was catalase. Once the reagent, tetramethyl-para-phenylenediamine dihydrochloride, was added
it appeared to portray characteristics of a positive reaction due to the bubbles forming on the
single line containing the organism; however, the initial observation did not correspond to the
appropriate end result pertaining to this organism. Consequently, 5 additional drops of hydrogen
peroxide was added to the organism where a closer examination of the media revealed small, yet
visible, bubbles created on the surface of the agar.
Discussion/Conclusion
Once the evaluation process is complete, the test results are then compiled and an overall
interpretation is documented. According to the assessment of the recorded results, as well as a
thorough investigation of the organism, unknown bacterium #02 portrayed all the necessary
characteristics for proper identification of the bacterium known as Pseudomonas aeruginosa.
All the tests performed on this organism resulted in a positive reaction, with the exception of
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Investigation of Unknown Bacterium #02
gelatin/protein hydrolysis and urea hydrolysis. Also, the Gram stain performed on the unknown
bacterium depicted the correct end result of a Gram negative smear. All these characteristics
combined resulted in the correct identification of the bacterium P. aeruginosa.
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Investigation of Unknown Bacterium #02
References
Acharya, Tankeshwar (2013). "Biochemical Tests Used to Identify Bacteria in
Microbiology Laboratory." Microbeonline Online Microbiology Guide.
Bauman, Robert W. (2009). Microbiology: With Diseases by Body System. 3rd ed. San
Francisco: Pearson Benjamin Cummings.
Centers for Disease Control and Prevention (2013). Healthcare-associated Infections
(HAIs). Centers for Disease Control and Prevention.
Eastfield College Microbiology Lab (2014). [Comparison Chart of Biochemical Test
Results]. Unpublished raw data.
Oliver, Tammy, Dr. (2013). Eastfield College Microbiology Biol 2420 Laboratory
Manual. 1st ed. Dubuque: Kendall Hunt.
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