Unknown #60
Proteus vulgaris
Maily Duque
07.29.26
Microbiology Lab
INTRODUCTION
The purpose of this experiment was to identify an unknown bacterium by examining its
morphological and biochemical characteristics. A series of laboratory tests were
performed to observe how the unknown organism responded under different conditions
and to determine its metabolic properties. The results from each test were analyzed
together to distinguish the unknown from other bacterial species. Through these
observations and test results, the unknown bacterium #60 was identified as Proteus
vulgaris.
Background Information
Proteus vulgaris is a Gram-negative bacillus bacterium that belongs to the
Morganellaceae family. It can be found in many environments including soil, water,
sewage, decaying organic material and it may also live in the intestinal tract of humans
and animals. Because it is a facultative anaerobe, P. vulgaris can grow whether oxygen is
present or not. One characteristic that makes this bacterium stand out is its high level of
motility. When grown on certain solid media, the cells can move together across the
surface and produce a rapid spreading pattern known as swarming.
Although P. vulgaris can exist without causing disease, it can become an opportunistic
pathogen when it reaches areas of the body where it would not normally be found. It has
been associated with urinary tract infections and wound infections. It produces urease,
an enzyme that breaks down urea and raises the pH of urine. This can contribute to
urinary stones during an infection. These characteristics are useful when identifying
Proteus vulgaris in the lab. Its motility and biochemical reactions help distinguish it from
other bacteria that may share similar features.
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MATERIALS
1.​ General Materials: Unknown bacterial culture #60, sterile inoculating loop, flint
striker, Bunsen burner, test tubes, test tube rack, microscope slides, compound
light microscope, immersion oil, distilled water bottle, Quebec colony counter and
incubator.
2.​ Gram Stain Reagents: Crystal violet, Gram's iodine, decolorizing alcohol, and
safranin.
3.​ Tube Media: Urea broth, SIM medium, tryptone broth, phenol red dextrose broth
with Durham tube, phenol red lactose broth with Durham tube, phenol red
sucrose broth with Durham tube, litmus milk, nutrient gelatin, nitrate broth,
MR-VP broth, peptone iron agar, phenylalanine agar, TSIA, Simmons citrate agar,
and thioglycollate medium.
4.​ Plate Media: PEA agar, MSA, EMB agar, DNase agar, Nutrient agar and Spirit Blue
agar.
5.​ Additional Reagents: Kovac's reagent, nitrate reagents A and B, zinc, methyl red,
alpha-naphthol, potassium hydroxide, ferric chloride, iodine, hydrogen peroxide,
and oxidase reagent.
Methods
Gram Stain
A small amount of Unknown #60 was transferred onto a clean microscope slide using a
sterile inoculating loop and mixed with a drop of distilled water to create a thin smear.
The smear was allowed to air dry and was then heat fixed by passing the slide over the
flame of the Bunsen burner. Crystal violet was applied to the smear for a minute and
then rinsed with distilled water. Gram's iodine was added for a minute and rinsed off,
followed by alcohol to decolorize the cells. The slide was rinsed again, and safranin was
applied as the counterstain for 3 minutes. After the final rinse, the slide was patted dry
carefully. Immersion oil was then added, and the slide was examined under the
microscope using the 100× oil immersion objective.
Urea Broth
Unknown #60 was transferred into the urea broth using a sterile inoculating loop. After
inoculation, the tube was gently swirled to mix the organism throughout the broth and
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was then incubated for approximately 24 hours. Following incubation, the broth was
observed for a color change. No additional reagent was added before the result was
observed.
Sulfide Indole Motility (SIM) Medium
Unknown #60 was inoculated into Sulfide Indole Motility (SIM) medium by making a
single straight slab into the agar with the Inoculating loop. The loop was withdrawn
along the same path without mixing the medium. The SIM tube was incubated for about
24 hours and then observed for blackening and for growth spreading away from the stab
line. Kovacs reagent was then added to the top of the medium and the top layer was
observed for a color change.
Tryptone Broth
Unknown #60 was transferred into the tryptone broth using a sterile inoculating loop.
The tube was gently mixed and incubated for approximately 24 hours. After incubation,
Kovacs reagent was added and the top layer of the broth was observed for color change.
Phenol Red Carbohydrate Broths
Three separate phenol red broths containing dextrose, lactose and sucrose were
inoculated with Unknown #60 using a sterile inoculating loop. Each broth was gently
mixed after inoculation and incubated for 24 hours. After incubation, the color of each
broth was observed and the Durham tubes were examined for trapped gas.
Litmus Milk
Unknown #60 was transferred into the litmus milk using a sterile inoculating loop. The
tube was gently mixed and incubated. After incubation, the medium was examined for
changes in color, consistency and appearance throughout the tube.
Nutrient Gelatin
Unknown #60 was inoculated into the nutrient gelatin and refrigerated for about 30
minutes. The tube was then removed from refrigeration and the consistency of the
medium was observed.
Nitrate Reduction
Unknown #60 was inoculated into nitrate broth and incubated for approximately 24 hours. After
incubation, nitrate reagents A and B were added and the tube was observed for a color change. If
no color developed after the addition of the two reagents, zinc was added and the tube was
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observed again.
Methyl Red and Voges-Proskauer (MR-VP) Tests
Unknown #60 was inoculated into Methyl Red and Voges- Proskauer (MR-VP) broth and
incubated. After incubation, the culture was used to perform the two tests. Methyl red
reagent was added for the MR test and the resulting color was observed. For the VP test,
alpha-naphitol and potassium hydroxide were added and then observed for a color
reaction.
Peptone Iron Agar
Unknown #60 was inoculated into peptone iron agar by making a straight slab into the
medium. The loop was withdrawn along the same path without mixing or streaking the
medium. The tube was incubated and later examined for changes in the appearances of
the agar.
Phenylalanine Agar
Unknown #60 was inoculated onto the phenylalanine agar slant by streaking the
organism across the slanted surface in a zig-zag pattern. The tube was incubated and
ferric chloride was added after incubation. The slant was then observed for a color
change.
Triple Sugar Iron Agar (TSIA)
Unknown #60 was inoculated into Triple Sugar Iron Agar (TSIA) by stabbing into the butt
of the agar and then streaking the slanted surface. The TSIA tube was incubated for
approximately 24 hours. After incubation, the slant and butt were observed for color
changes. The agar was also examined for blackening, cracks, bubbles or lifting.
Simmons Citrate Agar
Unknown #60 was inoculated onto the surface of the Simmons citrate agar slant by
streaking the organism across the slanted surface. The tube was incubated for
approximately 24 hours and then examined for bacterial growth and a change in the
color of the medium.
Thioglycollate Medium
Unknown #60 was inoculated into the thioglycollate medium and incubated. After incubation, the
location and pattern of bacterial growth throughout the tube were observed.
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Plate Tests- Methods
Phenylethyl Alcohol Agar (PEA)
Unknown #60 was streaked onto a Phenylethyl Alcohol Agar (PEA) plate using a sterile
inoculating loop. The plate was incubated and later examined using a Quebec colony
counter for bacterial growth.
Mannitol Salt Agar (MSA)
Unknown #60 was streaked onto a Mannitol Salt Agar (MSA) plate using a sterile
inoculating loop. The plate was incubated and later examined using a Quebec colony
counter for bacterial growth and changes in the appearance of the medium.
Eosin Methylene Blue (EMB) Agar
Unknown #60 was streaked onto an Eosin Methylene Blue (EMB) agar plate using a
sterile inoculating loop. After incubation, the plate was examined using a Quebec colony
counter for bacterial growth and changes in colony appearance.
Deoxyribonuclease (DNase) Agar
Unknown #60 was streaked onto a Deoxyribonuclease (DNase) agar plate using a sterile
inoculating loop. The plate was incubated and later examined using a Quebec colony
counter for changes surrounding the bacterial growth.
Spirit Blue Agar
Unknown #60 was streaked onto a Spirit Blue agar plate using a sterile inoculating loop.
After incubation, the bacterial growth and the surrounding medium were examined
using a Quebec colony counter for changes.
Catalase Test
Bacterial growth from Unknown #60 on a Nutrient Agar plate was used to perform the
catalase test. Hydrogen peroxide was applied to the bacterial growth, and the reaction
was observed immediately for bubble formation.
Oxidase Test
Bacterial growth from Unknown #60 on an Nutrient Agar plate was exposed to oxidase
reagents. The reaction was observed within approximately 20 seconds for a color
change.
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DATA:
Test
Observations
Urea Broth
Results
Urease positive (+)
-Broth turned hot pink
Thioglycollate Broth
Facultative anaerobe
-Cloudiness throughout the
broth, with greater
cloudiness toward the
bottom
Tryptone Broth
Indole negative (-)
-No pink/red layer formed
at the top after Kovacs
reagent was added.
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H₂S production negative
(-)
Peptone Iron Agar
-No blackening was
observed in the medium
Phenylalanine deaminase
positive (+)
Phenylalanine Agar
-Green color was present
after ferric chloride was
added
H₂S production positive
(+)
Sulfide Indole Motility
(SIM)
Indole positive (+)
-Blackening was observed
in the medium
-Pink layer formed at the
top after the Kovacs
reagent was added
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Motility positive (+)
-Growth spread away from
stab line
Nitrate reduction positive
(+)
Nitrate Reduction
-Broth turned red
immediately after nitrate
reagents A and B were
added.
Simmons Citrate Agar
Citrate negative (-)
-Medium remained green
Voges-Proskauer (VP)
VP negative(-)
-Bubbly pink/gray
appearance was observed
at the top, but no positive
red reaction happened
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Methyl Red (MR)
MR positive (+)
-Red color developed after
methyl red reagent was
added.
A/A
Gas negative (-)
Triple Sugar Iron Agar
(TSIA)
H₂S negative (-)
-Acidic slant and acidic
butt. No cracks/lifting or
blackening
Acid production negative(-)
Litmus reduction
positive(+)
Litmus Milk
Alkaline reaction positive
(+)
-No pink acid reaction
-white area at the bottom
-Purple and white
-White thick curd
-Purple stayed liquid at top.
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Peptonization negative (-)
Nutrient Gelatin
Gelatinase negative(-)
-Medium was solid after
refrigeration
Fermentation negative(-)
Phenol Red Lactose
Gas positive (+)
-Broth stayed red
-Gas was observed
Fermentation positive(+)
Phenol Red Sucrose
Gas positive (+)
-Broth turned yellow
-Gas was observed
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Fermentation positive(+)
Phenol Red Dextrose
Gas negative(-)
-Broth turned yellow
-No gas was observed
-Pink/red rod-shaped
cells were observed
under ben the
microscope
Gram-Stain
Gram-negative (-)
Plate Test Results:
Test
Observation
Result
Phenyl Ethyl Alcohol Agar
(PEA)
-No growth observed
Growth negative (-)
Mannitol Salt Agar
-No growth observed
-Medium remained yellow
Growth negative (-)
Mannitol fermentation
negative(-)
Eosin Methylene Blue Agar
-Growth was present
without a
lactose-fermentation color
change
Growth positive (+)
Lactose fermentation (-)
DNase Agar
-Heavy Growth with a visible DNase positive (+)
zone around the growth
Spirit Blue Agar
-Heavy growth but no lipid
hydrolysis reaction was
observed
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Lipase negative(-)
Catalase Test
-Immediate bubbling after
hydrogen peroxide was
added
Catalase positive(+)
Oxidase Test
-No dark purple/black color
developed within 20
seconds
Oxidase negative(-)
Discussion
The biochemical tests performed on Unknown #60 examined different
metabolic and enzymatic characteristics of the organism. Each test was
interpreted by comparing the observed reaction with the positive and negative
reactions for that test. When considered together, the results were used to
identify Unknown #60 as Proteus vulgaris.
The urea broth test determines whether an organism produces urease, an
enzyme that breaks down urea and produces ammonia. The ammonia raises the
pH of the medium. A positive reaction causes the phenol red indicator to become
bright pink, while a negative reaction does not produce this alkaline pink color.
Unknown #60 turned hot pink, indicating that it was urease positive.
The tryptone broth test determines whether an organism produces
tryptophanase, which breaks down tryptophan and produces indole. Kovac's
reagent is added after incubation to detect indole. A positive reaction forms a red
or pink layer at the top, while a negative reaction does not form this layer.
Unknown #60 was indole negative in the tryptone broth.
The nutrient gelatin test detects gelatinase, an extracellular enzyme that
hydrolyzes gelatin. A positive test remains liquid after refrigeration because the
gelatin has been broken down, while a negative test becomes solid again when
cooled. The medium for Unknown #60 was solid after refrigeration, indicating a
gelatinase-negative result.
The SIM test examines hydrogen sulfide production, indole production, and
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motility. H₂S production is positive when black precipitate forms and negative
when no blackening occurs. Unknown #60 showed blackening, making it H₂S
positive. Indole production is detected after Kovac's reagent is added. Formation
of a red or pink layer indicates a positive reaction, while the absence of this layer
is negative. Unknown #60 was indole positive in SIM. Motility is positive when
growth spreads away from the stab line and negative when growth remains
restricted to the stab line. Growth spread through the medium, indicating that the
organism was motile.
The peptone iron agar test also detects hydrogen sulfide production. A
positive reaction produces blackening at the stab line, while a negative reaction
does not. Unknown #60 showed no blackening and was therefore H₂S negative in
peptone iron agar. This differed from the positive H₂S result obtained with SIM.
The phenylalanine agar test determines whether an organism produces
phenylalanine deaminase, which removes an amino group from phenylalanine
and produces phenylpyruvic acid. Ferric chloride is added after incubation to
detect the product. Development of a green color indicates a positive reaction,
while the absence of green indicates a negative reaction. Unknown #60 developed
a green color and was phenylalanine deaminase positive.
The phenol red carbohydrate tests determine whether an organism can
ferment a specific carbohydrate and produce acid. Acid production lowers the pH
and causes the phenol red indicator to turn yellow, while a red or more alkaline
appearance indicates that fermentation was not detected. Gas production can
also be observed separately. Unknown #60 fermented dextrose and sucrose,
which produced yellow reactions, while lactose fermentation was negative
because the lactose broth remained red. Gas was negative in the dextrose tube
and positive in the lactose and sucrose tubes. The presence of gas in the lactose
tube despite the negative fermentation reaction was an inconsistent result and
may have resulted from an issue with the tube or interpretation of the reaction.
The litmus milk test can detect several metabolic reactions involving
lactose and milk proteins. Acid production causes the medium to become pink,
while an alkaline reaction causes a more purple appearance. Reduction of the
litmus indicator produces a white area, and peptonization involves breakdown of
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milk proteins and can cause the medium to become more translucent or clear.
Unknown #60 did not show the pink reaction expected for acid production,
making acid production negative. A white area indicated positive litmus
reduction, and the purple appearance indicated a positive alkaline reaction. The
medium remained thick rather than becoming clear, so peptonization was
negative.
The nitrate reduction test determines whether an organism can reduce
nitrate. Reagents A and B are added after incubation to detect nitrite.
Development of a red color immediately after these reagents are added indicates
that nitrate was reduced to nitrite and is a positive result. If no color develops,
the test requires an additional step before it can be interpreted as negative
because the nitrate may have been reduced beyond nitrite. Unknown #60 turned
red immediately after reagents A and B were added, indicating positive nitrate
reduction.
The Methyl Red (MR) and Voges-Proskauer (VP) tests examine different
pathways of glucose fermentation. The MR test detects stable acidic end products
produced through mixed acid fermentation. A red reaction after methyl red
reagent is added is positive, while a yellow reaction is negative. Unknown #60
was MR positive, indicating mixed acid fermentation. The VP test detects acetoin,
which is associated with the 2,3-butanediol fermentation pathway. Development
of the expected red reaction after the VP reagents are added indicates a positive
result, while failure to develop this reaction is negative. Unknown #60 was VP
negative.
The Triple Sugar Iron Agar (TSIA) test evaluates carbohydrate
fermentation, gas production, and H₂S production. A yellow acidic slant and
yellow acidic butt, or A/A, indicate fermentation sufficient to keep both portions
acidic. An alkaline red slant with an acidic yellow butt, or K/A, indicates glucose
fermentation without continued acid production throughout the slant. Gas
production can cause cracks, bubbles, or lifting of the agar, while H₂S production
causes blackening. Unknown #60 had an acidic slant and acidic butt (A/A) with no
evidence of gas production or blackening, making gas and H₂S negative.
The Simmons citrate test determines whether an organism can utilize
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citrate as its carbon source. Citrate utilization produces alkaline products,
causing the bromothymol blue indicator to change from green to blue. Growth
with a blue reaction indicates a positive result, while a medium that remains
green without citrate utilization is negative. Unknown #60 remained green and
was therefore citrate negative.
The thioglycollate broth test examines an organism's relationship with
oxygen by creating different oxygen concentrations throughout the tube. An
obligate aerobe grows mainly near the top where oxygen is highest, while an
obligate anaerobe grows toward the bottom where oxygen is limited. A
facultative anaerobe can grow throughout the tube because it can grow with or
without oxygen. Unknown #60 showed cloudiness throughout the broth and was
recorded as a facultative anaerobe.
The Phenyl Ethyl Alcohol (PEA) agar is selective because it favors the
growth of Gram-positive organisms while inhibiting many Gram-negative
organisms. Growth indicates that the organism can tolerate the selective
conditions, while no growth indicates inhibition. Unknown #60 showed no
growth on PEA, which was consistent with the Gram-negative result obtained
from the Gram stain.
The Mannitol Salt Agar (MSA) contains a high concentration of salt, making
it selective for organisms that can tolerate the salty environment. Growth
indicates salt tolerance, while no growth indicates that the organism was
inhibited. If growth occurs, mannitol fermentation can then be evaluated by a
yellow color change. Unknown #60 showed no growth, so it was not salt tolerant
under the conditions of the test, and mannitol fermentation could not be
determined from the plate.
The Eosin Methylene Blue (EMB) agar is selective for Gram-negative
organisms and differential for lactose fermentation. Growth supports the ability
to grow under the selective conditions, while lactose-fermenting organisms
produce characteristic darker coloration compared with nonfermenters.
Unknown #60 showed growth but no lactose-fermentation reaction, supporting
growth as a Gram-negative organism while indicating lactose fermentation was
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negative.
The DNase agar test detects DNase, an extracellular enzyme that
hydrolyzes DNA. A positive test produces the characteristic change or clearing
around bacterial growth, while a negative organism grows without this
surrounding reaction. Unknown #60 showed the reaction around the growth and
was therefore DNase positive.
The Spirit Blue agar test determines whether an organism produces lipase,
an extracellular enzyme that hydrolyzes lipids. A positive reaction produces the
characteristic change associated with lipid breakdown around the growth, while
a negative test shows growth without evidence of lipid hydrolysis. Unknown #60
grew on the plate but did not show lipid hydrolysis, indicating a lipase-negative
result.
The catalase test determines whether an organism produces catalase, an
enzyme that breaks hydrogen peroxide into water and oxygen. A positive
reaction produces fast bubbling after hydrogen peroxide is added because
oxygen gas is released, while a negative reaction produces no bubbles. Unknown
#60 bubbled immediately and was catalase positive.
The oxidase test detects cytochrome c oxidase, an enzyme involved in the
electron transport chain. A positive reaction produces a dark purple color , while
the absence of this color is negative. Unknown #60 did not produce the positive
reaction within 20 seconds and was therefore oxidase negative.
Overall, the results supported the identification of Unknown #60 as Proteus
vulgaris. Some results were inconsistent, including the indole and H₂S reactions
between different tests and gas production in the lactose tube despite negative
fermentation. These differences may have resulted from variations in the
procedure or interpretation of the reactions. The results were recorded as
observed rather than changed to match the expected characteristics of P. vulgaris.
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Conclusion
The purpose of this experiment was to identify Unknown #60 using staining, selective and differential
media, and biochemical tests. Based on the results, Unknown #60 was identified as Proteus vulgaris.
Although several unexpected reactions occurred, the combined characteristics of the unknown supported
the final identification.
REFERENCES
Drzewiecka, D. (2016). Significance and roles of Proteus spp. bacteria in natural
environments. Microbial Ecology, 72, 741–758.
National Center for Biotechnology Information. (n.d.). Proteus vulgaris. NCBI Taxonomy.
Supplemental Guide For Students In The General Microbiology Lab.
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Concept Maps:
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