Rachel Anthony

May 6th, 2019

4th Hour
Conclusion

Claim: The identity of unknown bacteria #11 is ​Citrobacter freundii.​

Evidence and Reasoning:

Initial Observations (Page 4): The snap tube displayed a cloudy appearance which supports the
claim that unknown #11 is ​Citrobacter freundii ​because the nutrient broth of the specimen is
opaque. Therefore, the similarity in the broth’s opacity support the claim that this bacteria is
Citrobacter freundii ​since they share the common characteristic, and the opacity of the solution
was the only characteristic of the bacteria that was able to be gathered from the initial
observations. An error that was present in the initial observations was specificalities of the broth
such as the color and observations of bacteria fragments were unable to be viewed clearly
because the snaptube was colored and opaque. Hence, the results that were gathered were
affected since the visual observations that were made regarding the opacity were hindered
because the snaptube impeded a clear visual of the bacteria.

Optimal Temperature (Pages 4-7): For 24, 48, and 72 hours, the plates at 30 degrees Celsius and
37 degrees Celsius experiences the largest quantity of growth. ​Citrobacter freundii ​optimal
growth temperature is between 30 degrees Celsius and 37 degrees Celsius which is why the
largest amount of growth appeared on the plates at 30 and 37 degrees Celsius. The two plates at
the different temperatures almost included the same quantity of growth, so it was challenging to
decide whether 30 degrees Celsius or 37 degrees Celsius is the optimal growth temperature;
however, after carefully examining the plate, it was concluded that 30 degrees Celsius was the
most suitable incubator temperature. Moreover, the shape of the unknown bacteria colonies and
that of ​Citrobacter freundii ​were both round and cream colored, which supports the claim that
unknown #11 is ​Citrobacter freundii ​since they share a common colony shape and color. An
error found in the optimal temperature test included putting a different amount of bacteria on
each inoculating loop. The variations in the quantity of bacteria placed on the nutrient agar
plates will differ how much bacteria will grow on each plate since the initial amount of it is not
the same. This could affect how the nutrient agar plates are interpreted because it could be
falsely pointing to a different optimal temperature, solely because there was more bacteria
originally on the plate.

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Gram stain (Page 8, 24): The gram stain displayed single coccobacillus specimen that had a size
of 2.0 by 0.5 microns. These are characteristics of ​Citrobacter freundii ​in addition to both
bacteria being-gram negative. Consequently, it was determined that unknown #11 is ​Citrobacter
freundii ​since their morphologies were identical. Possible errors in the gram stain include
inoculating too thick of a sample so that individual bacterium are not visible, which could lead to
an inaccurate determination of the arrangement and size, and using too much of the decolorizing
agent, ethanol, which could result in a falsely determined gram negative bacteria. However,
multiple grams stains were performed during the lab so the shape, size, arrangement, and type of
bacteria could be accurately determined without errors.

Catalase test (Page 9): The catalase test was positive for unknown #11 as indicated by the
production of gas bubbles. ​Citrobacter freundii ​also is catalase positive so this test supports the
claim that this unknown bateria’s identity is ​Citrobacter freundii. ​ An error present in the
catalase test would be that the inoculated bacteria is too old because older bacteria might not
display that the bacteria produces catalase. However, unknown #11 displayed that it produced
catalase, so it can be determined that a young enough colony was used in this test.

Nutrient broth (Page 11): ​Citrobacter freundii ​is a facultative anaerobe which explains why
there was bacterial growth throughout the entire test tube. The tube had a turbid appearance with
a large viscid sediment, which adds strength to the argument that unknown #11 is ​Citrobacter
freundii ​since facultative anaerobes display growth all throughout the test tube which was
observed. The main error in the nutrient broth test is that sterile conditions were not maintained
throughout the entire process whether this was through failing to create a hot pocket of air at the
opening of the test tube or improperly sterilizing an inoculating loop and letting it cool. This
could lead to contamination of the test tube which could caused different specimens to grow in
other areas of the test tube, providing inaccurate data on the oxygen requirements of the bacteria.

Motility hanging drop test (Page 13): No motion was observed in the hanging drop test, so it can
be concluded that unknown #11 does not have any flagella. ​Citrobacter freundii ​most often has
several flagella but there are some bacteria that are non-motile; consequently, since no motile
specimens were viewed and some bacteria of ​Citrobacter freundii ​are non-motile, it can be
concluded that unknown #11 is ​Citrobacter freundii.​ One error present in the hanging drop
motility test was that the broth used to test for motility did not contain a large amount of bacteria.
Another error could be that the sample was not viewed at the position of the edge of the nutrient
broth dot because that is where locomotion would be observed. Due to these errors, the bacterial
unknown could have displayed motility but it did not.

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Nutrient agar slant (Pages 12 and 21): The nutrient agar slant was translucent with an echinulate
growth pattern. The cream color and the moderate amount of growth aid the argument that
unknown #11 is ​Citrobacter freundii ​since they both display a cream color and they both grew
well in the optimal temperature. An error possible in the nutrient agar slant test is that the
inoculating loop touched the test tube before it was dragged down. This would cause a larger
quantity of the bacteria to be on the tube which would leave a little amount that would grow on
the nutrient agar.

Starch agar and hydrolysis test (Page 13 and 15): The cream colored bacterium does not show
indication that it hydrolyzes starch as determined by the absence of a clear halo forming around
the bacteria after Gram’s iodine was poured over it. Unknown #11 and ​Citrobacter freundii ​both
do not hydrolyze starch which is a component to why it was determined that ​Citrobacter freundii
is the identity of the unknown. An error with the starch hydrolysis test could be that the Gram’s
iodine was not left on the bacteria for a long enough period of time because the rate of hydrolysis
varies for bacteria so it could produce a false negative if enough time was not given to provide
correct results.

Gelatin stab (Page 17): The gelatin had crateriform growth with no hydrolysis of gelatin.
Crateriform growth suggests that the bacteria either needs or prefers a source of oxygen, and
since ​Citrobacter freundii i​ s a facultative anaerobe, the growth pattern explains why ​Citrobacter
freundii ​is the concluded identity of the unknown since they both prefer oxygen. Furthermore,
both unknown #11 and ​Citrobacter freundii ​do not hydrolyze gelatin which further justifies the
claim. An error present in the gelatin stab test is that the test tube was placed in the ice bath for
too long because it would cause the gelatin to freeze and be negative for hydrolysis when it could
actually be positive. However, this is why the gelatin test tube was only placed in an ice bath for
five minutes so that it would not freeze and provide unreliable data.

Potato agar and hydrolysis (Page 20): After Gram’s iodine was placed on the potato agar plate,
no halo appeared around the bacteria; consequently, unknown #11 does not hydrolyze the starch.
This defends the claim that this unknown is ​Citrobacter freundii ​since they both do not
hydrolyze starch. An error in the potato starch hydrolysis test was that the Gram’s iodine was
not given enough time to display the hydrolysis of the starch which would cause could provide a
false result that the bacteria does not hydrolyze the starch.

Phenol red dextrose (Page 19): The dextrose tube showed signs of fermentation of dextrose as
revealed by the change in color to a bright yellow, and it also showed a production of a gas in the
Durham tube. Acid and gas are produced from dextrose by ​Citrobacter freundii ​which is a
reason why it was concluded unknown #11 is ​Citrobacter freundii ​since they share this common
characteristic. Phenol red tubes can change based on the environment that they are in so that is a

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possible error in the test. Also another error would be that there is already some gas present in
the Durham tube because it would influence the results by giving an automatically positive test
for the production of gas from dextrose. Because of these possible errors, the phenol red
dextrose tube was compared to a control tube in order to provide the most accurate results
possible.

Phenol red lactose (Page 19): Acid and gas were produced from the unknown in the lactose tube
because the phenol red liquid turned a yellow color and there was a gas bubble in the Durham
tube. ​Citrobacter freundii ​is positive for acid and gas production from lactose; hence, unknown
#11 is ​Citrobacter freundii ​since there is production of acid and gas from lactose in both of them.
An error in the phenol red lactose tube was the temperature change caused a change in the color
and not necessarily the bacteria present; however, this error was avoided by having a set of
controls used to compare the tubes.

Phenol red sucrose (Page 19): The phenol red sucrose tube showed sign of some acid production
because the test tube turned light orange, but there was no gas produced from the tube. Although
Citrobacter freundii ​produces both acid and almost always gas from sucrose, the error in no gas
production can be attributed to the amount of time the tube was observed because some bacteria
require more time to produce it than others. The sucrose tube should have been placed into the
incubator for another day, but it was only placed in there for 24 hours. Consequently, the data
received could be inaccurate since it needed more time to show production of the gas;
nevertheless, the similarity in the acid production from sucrose for unknown #11 and ​Citrobacter
freundii ​enhances the argument that they are the same bacteria.

Colony identification (Pages 22 and 23): Unknown #11 has round, glossy colonies with convex
elevations and wavy margins. ​Citrobacter freundii ​has round, glistening colonies with convex
elevations and irregular margins. The discrepancy between the wavy and irregular margins can
be attributed to the fact that the devices that were used to observe the colonies did not provide
extremely clear images of the colonies. However, colonies with irregular margins have a wavy
appearance it is just not as uniform. Besides the margins, unknown #11 and ​Citrobacter freundii
share common colony characteristics which defends the conclusion that it is the identity of the
unknown. As noted before, the main error in the colony identification is that the tools used to
examine the bacteria did not necessarily provide the most accurate information since they did not
provide clear and extremely magnified images of the individual colonies.

EMB agar (Page 25 and 27): Extensive dark purple growth was observed on EMB agar which
indicates fermentation of lactose. ​Citrobacter freundii c​ an either exhibit a dark purple color on
EMB agar or a metallic green, depending on the fermentation abilities of the individual bacterial
strains. Because the EMB agar displayed a dark purple color, the claim that ​Citrobacter freundii

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is the identity of unknown #11 is supported by this evidence. An error with the EMB agar would
be that growth of the bacteria is present when it is actually just the mark made when the bacteria
was inoculated onto the agar. Due to this possible error, it was decided that the agar would be
placed into the incubator for an additional 24 hours to provide a clear confirmation of growth,
which it did.

Skim milk agar: Skim milk agar was not used in tests for unknown #11 because other tests were
being performed that provided more conclusive results of what the identity of the bacterial
unknown is. Also, this medium works effectively on gram-positive bacteria and so, due to the
bacteria being gram-negative, the unknown was not inoculated onto the skim milk agar.

Mannitol salt agar: Unknown #11 was not inoculated onto mannitol salt agar because it is a
differential medium that inhibits the growth of gram negative bacteria. It has a high
concentration of salt, and the gram-negative bacteria would not be able to ferment mannitol and
grow on the agar. Therefore, it was illogical to put the bacteria on the agar because the salt
concentration would cause the bacteria to not grow on the medium at all.

Spirit blue agar: Spirit blue agar is a differential agar for gram-positive rod bacteria. Because
unknown is #11 is gram-negative, the spirit blue agar will prevent the bacteria from growing on
the medium, so it will be indeterminate if the bacteria has the ability to hydrolyze the lipids.

MacConkey agar (Page 25 and 27): The unknown displayed a vibrant magenta color on
MacConkey agar which means that it is a strong fermenter of lactose. ​Citrobacter freundii
ferments lactose and also produces a vivid pink color on MacConkey agar, aiding the conclusion
that unknown #11 is ​Citrobacter freundii,​ since they both produce a characteristic extremely
bright pink color when placed on MacConkey agar. An error in this test would be believing the
bacteria ferments lactose due to a pink color production when in reality, the pink color was
caused from the bacteria being inoculated onto the agar.

Therefore, due to the plethora of commonalities between unknown #11 and ​Citrobacter freundii​,
it can be concluded that it is the identity of the unknown.

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Claim: The identity of unknown bacteria #38 is ​Bacillus brevis.​

Evidence and Reasoning:

Initial observations (Page 4): The bacterial broth had an opaque appearance to it in the test tube.
Bacillus brevis h​ as a high turbidity in broth so this observation supports the claim that unknown
#38 is ​Bacillus brevis ​since they both display a common opacity in broth. This was the only
observation gathered from the initial observation of the bacteria in the snaptube because the snap
tube was opaque. Thus, it could have been wrongly determined that the bacteria had a opaque
appearance because the snap tube was opaque as well. Also, a definitive color was not able to be
determined clearly through the snap tube since it was yellow.

Optimal temperature (Pages 4-7): Unknown #38 had very little growth on the plate incubated at
25 degrees Celsius during the 72 hour period of growth on the nutrient agar, while the plates at
30 degrees Celsius and 37 degrees Celsius experienced the largest quantity of growth. ​Bacillus
brevis ​has an optimal growth temperature between 30 degrees Celsius and 37 degrees Celsius
which is why the data supports the conclusion that unknown #38 is ​Bacillus brevis ​since the
unknown grew the most in the optimal temperature range of ​Bacillus brevis ​and grew minimally
at the temperature outside of its temperature range. However, the differences in the amount of
growth present could be attributed to the different amount of bacteria that was inoculated onto
the plate and the location of where it was inoculated. If the nutrient plate started out with a
smaller amount of bacteria, it would take longer for more bacteria to grow and cause the
conclusion to be drawn that it does not grow best at that temperature. Hence, this is why the
bacteria was observed for 72 hours to see the growth patterns over a longer period of time.

Gram stain (Page 8): The gram stain determined that unknown #38 was gram-positive and single
bacillus with a size of 8.0 by 2.0 microns. ​Bacillus brevis i​ s found in singles, gram-positive, and
has an average size of about 6.0 by 0.5 microns. Unknown #38 and ​Bacillus brevis ​share a
common shape and arrangement, and their sizes are very close to one another with the variation
being attributed to slight errors in measurement on the microscope. Subterminal spores were also
observed on the gram stain which are a characteristic of ​Bacillus brevis​; consequently, the
information gathered from the gram stain justify the argument that unknown #38 is bacillus
brevis since they share morphological features. An error in the gram stain process was too much
decolorizer was used which made the bacteria appear to be gram-negative. However, multiple
gram stains were performed in order to confidently establish that this unknown is gram-positive.

Catalase test (Page 9): No gas bubbles were produced when hydrogen peroxide was added to a
sample of unknown #38, so it does not produce catalase. ​Bacillus brevis i​ s also catalase
negative, but some bubbles may appear after a few minutes due to a slow reaction. Since

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unknown #38 and ​Bacillus brevis ​are both catalase negative, this test aids the argument that it is
the identity of the unknown. An error present in the catalase test is that the inoculated bacteria is
too old to produce a reaction with hydrogen peroxide, which would possibly give the results a
false negative. To reduce this error, a second catalase test was performed at the end of the lab
which proved that unknown #38 does not produce catalase.

Nutrient broth (Page 11): The nutrient broth test tube had ring surface growth, with granular
particles suspended in solution, and flocculent sediment. ​Bacillus brevis i​ s an aerobic bacterium
which explains why there was ring growth on the test tube because the bacterium requires large
amounts of oxygen. There were some bacterial particles suspended throughout the broth because
of the different requirements of the individual bacteria, but the majority of the particles were near
the top of the broth. Hence, due to the location of the bacteria at the top of the test tube, the
conclusion that unknown #38 is ​Bacillus brevis ​is defended. An error with the nutrient broth test
tube is that it became disturbed before it was analyzed because it could disrupt the location of the
bacteria so their oxygen requirements would be determined incorrectly since they would be
dispositioned.

Motility hanging drop test (Page 13): Unknown #38 was observed to be motile and displayed
Brownian motion. ​Bacillus brevis i​ s motile and so the observation made that there was
movement for the hanging drop test supports that it is the identity of unknown #38. An error
with the hanging drop test would be to mistake the movement of broth and crystal violet with
movement of the actual bacteria because the drop spreads out on the slide so it is essential that
the edge of the drop is observed to movement. Because of this possible error, a hanging drop test
was performed multiple times for confirmation of motility.

Nutrient agar slant (Pages 12 and 21): The agar slant growth was opaque, round and greasy with
a cream color and had beaded growth. ​Bacillus brevis h​ as an opaque appearance and the
individual colonies are round, so the beaded appearance and opaqueness of the agar slant growth
support the claim that unknown #38 is ​Bacillus brevis b​ ecause they both display a common
growth appearance on nutrient agar. An error in the nutrient agar test is that the inoculating loop
touched the bottom of the test tube which caused the bacteria to be on there and not have as
much growth on the agar slant. This would change the opacity since there would not be as much
growth on the slant and could possibly affect the form of the growth if the bacteria was not given
enough time to develop since there would be a minimal amount of it.

Starch agar and hydrolysis test (Pages 13 and 15): No hydrolysis of starch was present for
unknown #38 because there were no clear halos surrounding the bacterial colonies after Gram’s
iodine was left on it for a few minutes. ​Bacillus brevis a​ lso does not hydrolyze starch which
adds to the argument that it is the identity of unknown #38 since starch hydrolysis is not present

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in either bacteria. An error in the starch hydrolysis test is that the Gram’s iodine was not left on
the starch agar plate long enough so the bacteria could show digestion of the starch because the
hydrolysis rates vary between bacteria so the bacteria could have digested the starch but it just
required a longer period of time to show it.

Gelatin stab (Page 17): The gelatin stab experienced no liquefaction and there was minimal
growth throughout the gelatin. ​Bacillus brevis a​ lso does not liquify gelatin which is a supporting
piece of evidence that ​Bacillus brevis ​is the identity of unknown #38. An error in the gelatin stab
would be touching the inoculating loop to the bottom of the test tube because it would deposit a
large amount of the bacteria there, so the growth of the bacteria throughout the tube would not be
viewed since it would all be at the bottom of the tube. This would ultimately cause a distortion
of the form of the growth of the bacteria.

Potato agar and hydrolysis test (Page 20): Unknown #38 did not hydrolyze potato starch since
there was not clear halo that surrounded the bacteria that would suggest this occurred. Lack of
starch hydrolysis is also found with ​Bacillus brevis ​which is why this test supports the claim that
this unknown is ​Bacillus brevis. ​Again, an error with the potato hydrolysis test is that the
Gram’s iodine was not given enough time to hydrolyze the starch so it could provide a false
negative for the test because the rate of hydrolysis differs between bacteria.

Phenol red dextrose (Pages 19 and 21): The phenol red dextrose tube had a vibrant orange color
with no gas bubble, and when it was compared to the control dextrose tube for 37 degrees
Celsius, it was determined that the bacteria does not produce acid in dextrose. There was a slight
change in the color but it was not different enough to provide the conclusion that the unknown
bacteria produces acid in dextrose. ​Bacillus brevis d​ oes not produce acid or gas from dextrose so
there is another similarity between the two bacteria that supports the relationship between them.
An error with the phenol red tubes is that they can be affected based on the environment
surrounding them which is why is was essential that there were controls for each temperature so
that there would be something to compare the bacterial phenol red tubes with, or else false
positive or false negative results for acid formation could occur.

Phenol red lactose (Pages 19 and 21): The phenol red lactose tube had a light reddish-orange
color that was extremely similar to the control tube and had no gas production. ​Bacillus brevis
also does not ferment lactose or produce gas from it, leading to the conclusion that it is the
identity of the unknown. Another error present in the phenol tubes is that the tubes were not
given enough time to show signs of acid or gas production; because of this potential error, all
three phenol red tubes were observed for 48 hours before final conclusions were drawn to
confirm that there was no acid or gas production.

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Phenol red sucrose (Pages 19 and 21): Acid and gas production did not occur in the phenol red
sucrose tube for unknown #38. ​Bacillus brevis m ​ ay or may not ferment sucrose depending on
the bacterial strain; however, since the unknown did not ferment sucrose, the results still support
the claim that ​Bacillus brevis i​ s the identity of unknown #38. This test tube could have also
been impacted by its outside environment and the amount of time that it was observed because
they both could have altered the production of acid or gas in the test tube, but controls and a 48
hour observation period were used to try to minimize the possible errors.

Colony identification (Pages 22 and 23): Young colonies of unknown #38 have a round
appearance but as they get older, cells move from the center producing wavy margins. The
colonies also have more translucent border around the colonies with them becoming more
opaque towards the middle. Moreover, the colonies have a glossy appearance and a raised
elevation. ​Bacillus brevis a​ lso has round, opaque becoming translucent toward the edges
colonies which justifies the position that it is the identity of unknown #38 because their colonies’
appearances are extremely similar. An error with the colony observations is that the instruments
used to determine descriptions of the colonies did not provide clear visuals of the margins or
elevations which made it difficult to determine them. After careful observations, final
observations were made about those aspects of the colonies’ characteristics.

EMB agar: Unknown #38 was not inoculated onto EMB agar because it was already determined
that it could not ferment lactose and that is what EMB agar tests for. Also, EMB agar is a
selective medium that will inhibit the growth of gram-positive bacteria, so unknown #38 will not
even have the ability to grow on the medium, making it useless to test it.

Skim milk agar (Pages 25 and 27): Hydrolysis of casein was observed on the skim milk agar
because there were clear areas surrounding the bacterial growth on the agar. ​Bacillus brevis
hydrolyzes casein so this additional test provides more evidence that ​Bacillus brevis ​is the
identity of unknown #38. A possible error for the skim milk agar was that the bacteria was not
allowed to grow for a long enough period of time on the agar because the bacteria did grow very
lightly on the agar; however, after 48 hours, it was clear that there was signs of hydrolysis of
casein due to the clear halos surrounding the colonies.

Mannitol salt agar (Page 28): The mannitol salt agar remained bright orange after 24 hours and
displayed no visible growth of colonies. ​Bacillus brevis ​has minimal to no growth on mannitol
salt agar and does not illustrate a color change in the agar, so the observations support the theory
that unknown #38 is ​Bacillus brevis. ​ The bacteria was only kept on the mannitol salt agar for 24
hours due to time restraints; it would have been more thorough to incubate it for another 24
hours to determine if there was any slow change or grow in the agar which is a possible error in
this test.

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Spirit blue agar (Page 28): Spirit blue agar possesses hydrolysis of lipids because there was a
faint blue color surrounding the bacteria while a darker blue color remained on the other portions
of the plate where the bacteria was not present. ​Bacillus brevis i​ s also positive for hydrolysis of
lipids and has a light blue tint to it as well; hence, it is the identity of unknown #38 since they
portray the same growth and hydrolyzation for spirit blue agar. An error in spirit blue tests is
that it needs to be incubated for more than 24 hours to reveal hydrolysis of lipids, but the agar
clearly defined that hydrolysis was present after 24 hours so this error did not affected the
gathered results.

MacConkey agar: Unknown #38 was not tested on MacConkey agar because it is a selective
medium that only allows gram negative bacteria to grow on it, and it also tests for fermentation
of lactose which was already tested for in the phenol red lactose tube. Therefore, it would not be
reasonable to inoculate it since there would not be a chance that the bacteria would grow on it,
and a different test already provided information that the agar tests.

All in all, the extensive similarities that existed between the two bacteria and the minimal
discrepancies between them prove that ​Bacillus brevis i​ s the identity of unknown #38.

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