Create DICHOTOMOUS KEY showing which selective media and
biochemical tests you would need if you were to test your unknown
bacteria (Clostridium Botulinum) in the lab and
also, describe the dichotomous key of Clostridium
Botulinum in paragraph like in example shown
below.
[ Like this is example for Pseudomonas
aeruginosa shown by my professor:
According to my unknown bacterium 16S DNA sequence result from
NCBI database, my unknown bacterium was identified to be
Pseudomonas aeruginosa. In order to make sure I have
enough samples for the subsequent tests, I would grow the bacteria
in a nutrient broth. The first round of tests I would do would be
tests for physical characteristics in order to narrow down what
group my bacteria falls under in Bergey’s Manual. The first test I
would want to do would be to find the shape and arrangement. After
looking at the bacteria under the microscope, I would see that they
are rod shaped(Bacillus) and in a single arrangement. The next test
I would do is Gram Staining to determine if the bacteria is gram
positive or negative. After Gram Staining, I would notice the
bacteria is reddish in color. Since the crystal violet is washed
out and the Safranin counterstain colored the bacteria red, this
indicates that the bacteria is gram negative.
After testing the physical characteristics, I would test its
oxygen tolerance. To do this, I would test the bacteria in Fluid
Thioglycolate Medium(FTM). After observing the bacteria only grew
in the top layer, I would surmise that the bacteria needed oxygen
to survive, meaning it is an Obligate Aerobe.
From these facts, I could determine that the bacteria fit in
Group 4 in Bergey’s Manual. Key differences between the different
genera in this group include pigments/fluorescent, motility, growth
requirements, denitrification, morphology, oxidase.
After we know the bacteria is gram negative and a bacillus
structure, I would prepare tests with SIM media. The media contains
casein and animal tissue as sources for amino acids, an iron
containing compound, and sulfur in the form of sodium thiosulfate.
The first test I would do with this media is the Indole test which
is used to determine if the organism possesses the Tryptophanase
enzyme. The media is inoculated with a needle and tests indole
production from tryptophan in the casein and animal tissue, made
possible by the tryptophanase enzyme. After incubation, Kovac’s
reagent is added which forms a band at the top of the agar. Any
indole present will react with Kovac’s reagent, turning the strip
red. In our case, we would expect to see no color change as our
bacteria is Indole negative.
Following this, the Urease test will follow to see if the bacteria
possesses the urease enzyme in order to digest Urea. The media used
is a urea broth in a test tube, where the only source of nutrients
other than urea is a trace amount of yeast extract. Buffers are
also present in order to stop the alkalization of the media except
for rapid urease positive bacteria. Phenol red is used as pH
indicating dye. It is yellow to orange below pH 8.4 and red to pink
above. Orange or yellow is negative and red or pink is a positive
result. In my case, there is no urea hydrolysis, so the broth will
be yellow or orange, indicating a negative result.
Using SIM media again, I would perform the Sulfur(H2S)
test. This tests for sulfur reduction. In this media, when sulfur
is reduced, H2S gas will react with the iron in the
media. This will cause ferric sulfide to form, which is a black
precipitate. Since the media did not turn black, we can conclude
that the bacteria is H2S negative.
Lastly, I would perform a Casease test in order to test if the
bacteria could hydrolyze the protein found in milk. In this test,
we would use a milk agar which contains pancreatic digest of
casein, yeast extract, dextrose, and powdered milk. A clearing in
this milk agar on the plate means that the bacteria has the casease
enzyme and was able to digest the casein in the agar. In our case,
there would be a positive result as a clearing is observed.
Following Bergey’s Manual, this would narrow down the choices,
confirming Pseudomonas aeruginosa.
Like wise in the same way, as above example for Pseudomonas
aeruginosa,
1) Create DICHOTOMOUS KEY showing which selective media and
biochemical tests you would need if you were to test your unknown
bacteria (Clostridium Botulinum) in the lab and
2)Describe the dichotomous key of Clostridium
Botulinum in paragraph like in example shown.