Morgan McBurnie and Nick Rause 

Medical Microbiology Lab - SBL 219 02 

Dr. Reay 
Due: April 23, 2020 

Identification of a Bacterial Unknown: Using Structural and Biochemical Characteristics

as well as Biochemical Characteristics to Identify Serratia marcescens
Abstract: 
Identification of an unknown bacteria is very important in the healthcare field and
environmental studies when a bacteria of unknown origin is atypically found in a patient or
location other than human. Through tests and assays, the identity of an unknown bacteria was
identified. The tests conducted looked at both structural and biochemical characteristics of the
unknown bacteria and the bacteria was identified as S. marcescens. The results from the tests
were compared to documented and previously established characteristics of S. marcescens to
verify and guide the succeeding assays. 

Introduction: 
Identification of a bacterial unknown can be very helpful when a patient presents with an
illness that is suspected to be caused by a bacteria. A physician may want to identify the bacteria
in a patient’s blood to help guide the patient’s treatment plan (Pomerville 2018). Also, a drug
company may hire a microbiologist to identify a bacteria as a possible candidate for new
antibiotics (Pomerville 2018). Microbiologists play an important role in the identification of
bacteria for many other needs such as contamination of food and water, pollution of the
environment, and bacteria becoming drug resistant (Pomerville 2018, Kim 2020). An unknown
bacteria needs to be identified using assays to distinguish between structural and biochemical
characteristics of various bacteria. Identification in a timely and efficient manner is also
important because a patient may be in critical condition and the causative agent of their illness
needs to be identified quickly to allow for the best chance of recovery and survival. Different
assays allow the microbiologist to narrow down a causative agent of a particular illness. These
tests look at various components of the bacteria such as shape, presence or absence of a substrate
or product, and the enzymes associated with the bacteria (Pomerville 2018). Also, interpretation
of results can speed up the identification process by knowing what the result of another test
would be even before performing it, like the endospore test. For example, a bacteria that is
identified as a Gram-negative bacteria by using Gram-staining techniques leads a microbiologist
to acknowledge an endospore test does not need to be completed. Identifying a bacterial
unknown is again found using various structural and biochemical assays quickly and efficiently.
Gram-staining is an important differential testing method because the great majority of bacteria
are either Gram-negative or Gram-positive (Pomerville 2018). The results from the Gram-stain
differential test are used to assess the necessity of performing an endospore test to anticipate the
production of endospores. The appearance of the colony shape as well as the individual bacteria
shape identified on the agar plate and under a light microscope, respectively, are useful in
narrowing down the identity of specific unknown (Pomerville 2018). Growth on a thioglycolate
media under different temperatures, such as 25C and 37C, show the preference of the specific
bacterial unknown. Carbohydrate fermentation of various carbohydrate sources, such as glucose,
lactose, and sucrose, will conclude if a particular carbohydrate is fermented by a bacterial
unknown as indicated by a color change of the media (Pomerville 2018). This test concluded that
the bacterial unknown produced carbon dioxide as a product as well. A catalase assay was
performed to indicate the presence of the enzyme catalase by breakdown of hydrogen peroxide.
A SIM agar media showed three characteristics of an unknown bacteria like the production of
sulfide and indole production (Pomerville 2018). The other characteristic found in the SIM
media was motility. The production of a high concentration of organic acid is shown when a
methyl red indicator is added to an inoculated tube with a bacterial unknown in a media
containing glucose. Simmons citrate agar detects the ability of a bacterial unknown to use citrate
as a sole energy source (Pomerville 2018). The presence of lipase enzymes are indicated by the
breakdown of triglycerides in a fat (triglyceride) hydrolysis assay. Lastly, other enzymes, known
as proteases, are found in bacteria that break down proteins such as the enzyme casein which
would be found in a positive casein hydrolysis assay (Pomerville 2018). Using all of the assays
above, the identification of a bacterial unknown can be determined due to the results concluded
from the tests and compared to known cultural characteristics as well as “fingerprint”
biochemical characteristics that are specific to a bacteria in the environment. 
 
Material and Methods:
Gram-stain: A sterile field was created with a Bunsen Burner and a microscope slide was
obtained. A bacterial unknown was chosen. Using a fired, sterilized inoculating loop, a small
drop of distilled water was applied to the microscope slide. A small amount of unknown bacteria
was placed in the distilled water using a fired, sterile inoculating loop. The slide was let to air dry
under the sterile field. Once dry, the bacteria was heat-fixed into the slide by passing it over the
bunsen burner flame three times for one second each over the flame, using a clothespin tool.
Once the unknown bacteria was heat-fixed to the slide, a staining tray was obtained. The
microscope slide containing the bacterial unknown as placed on the rack in the staining tray. The
unknown bacterial smear was flooded with crystal violet stain (the primary stain). After one
minute, the stain was rinsed off the slide using distilled water and shook to remove excess water.
Next, the slide was flooded with Gram’s iodine. After one minute, the slide was rinsed with
distilled water and shook to remove excess water. The smear was then decolorized using 95%
ethyl alcohol, as a few drops were added to the smear. The drops were slid back and forth by
tilting the slide. The alcohol was allowed to drip off and the decolorizing step was repeated for a
second time. After the second decolorizing attempt, the slide was rinsed with distilled water and
shook to remove excess water. Next, safranin, a counterstain, was added to the smear. After 30
seconds, the smear was rinsed and blotted using blotting paper (bibulous paper). The smear was
viewed under a microscope to look for characteristics. 
Endospore: The endospore formation test was not needed due to the results obtained from
the previous Gram-stain test.
Pigmentation: A nutrient agar plate was prepared during the previous lab period. The
plate was divided into four quadrants. The unknown bacteria was obtained using a fired, sterile
inoculating loop under a Bunsen Burner sterile environment. The inoculating loop containing the
bacterial unknown was streaked into the first quadrant using streak plating techniques. The
inoculating loop was fired to sterilize. Next, the sterile inoculating loop was used to streak
bacterial unknown from the first quadrant into the second quadrant, making two to three passes
back into the first quadrant. The inoculating loop was fired. Next, the sterile inoculating loop was
used to streak bacterial unknown from the second quadrant to the third quadrant, making two to
three passes back into the second quadrant. Lastly, the inoculating loop was fired. The sterile
inoculating loop was used to streak bacterial unknown from the third quadrant into the fourth
quadrant, making two to three passes from the third quadrant into the fourth. The inoculated agar
plate was incubated at 30C for 48 hours and observed for pigmentation. 
Temperature preference: Two nutrient agar slates were prepared during the previous lab
and then obtained for the assay. The unknown bacteria was obtained using a fired, sterile
inoculating loop under a Bunsen Burner sterile environment. A small amount of bacterial
unknown was inoculated on one the agar slants that was incubated at 25C, labeled “25”. Next,
using a fired, sterile inoculating loop, another small amount of bacterial unknown was inoculated
onto the second agar slant that was incubated at 37C, labeled “37”. After incubation, the slants
were examined for growth. 
Oxygen Gas Requirement (Thioglycolate): A tube with thioglycolate medium was
obtained. The unknown bacteria was obtained. Using a fired, sterile inoculating loop under a
Bunsen Burner sterile environment, a small amount of bacterial unknown was inoculated into the
thioglycolate medium. The thioglycolate medium was rotated in the hands ten times. The tube
was incubated at room temperature for 24-48 hours. The tube was then observed for growth. 
Carbohydrate Fermentation and Gas production: Nine tubes of various carbohydrate
mediums containing a phenol red indicator were prepared during the previous lab and then were
obtained for the assay. Inside the tubes, a Durham tube was placed in the media invertedly so the
gas was trapped if produced. Three of the nine tubes contained media containing glucose media,
another three of the nine tubes containing media contained lactose media, and the last three tubes
containing media contained sucrose media. The tubes were labeled respectively with the type of
carbohydrate as well as the bacteria that was inoculated in them. A small amount of Escherichia
coli was obtained using a fired, sterile inoculating loop under a Bunsen Burner sterile
environment and inoculated into the tube labeled “Glucose and E. coli” containing the glucose
media and shaken lightly. Next, a fired, sterile inoculating loop was used to inoculate another
small amount of E. coli into the tube labeled “Lactose and E. coli” containing the lactose media
and shaken lightly. Next, a fired, sterile inoculating loop was used to inoculate another small
amount of E. coli into one of the tubes labeled “Sucrose and E. coli” containing the sucrose
media and shaken lightly. This process as stated before was performed again but instead of E.
coli, Micrococcus luteus was inoculated in three tubes, one containing the glucose media, one
containing the lactose media, and one containing the sucrose media, all labeled respectively.
Next, the process explained before was repeated once more but this time the bacterial unknown
was inoculated into one tube containing the glucose media, one tube containing the lactose
media, and one tube containing the sucrose media, all labeled respectively. The inoculated tubes
were incubated for 24 to 48 hours at 37C. After incubation, the media was examined for
fermentation as well as gas production. 
Catalase Assay: A nutrient agar plate was obtained. The plate was divided in half. The
one side of the plate was labeled as “unknown” and the opposite side was labeled as “E. coli”. A
small amount of the bacterial unknown was obtained using a fired, sterile inoculating loop and
inoculated into the nutrient agar plate on the side labeled “unknown”. Next, using a fired, sterile
inoculating loop, a small amount of E. coli was inoculated into the nutrient agar plate on the side
labeled “E. coli”. The plate containing the bacterial unknown and E. coli was incubated for 24 to
48 hours at 37C. After incubation, several drops of three percent hydrogen peroxide were
dropped directly onto the bacterial unknown and E. coli on the plate. Once the three percent
hydrogen peroxide was added, the plate was examined for the presence of bubble formation
(effervesce).
 SIM Assay: Three SIM (Sulfide Indole Motility) agar tubes were prepared in the previous
lab and then were obtained for this assay. The tubes were labeled as follows: “E. coli”,
“Citrobacter freundii”, and “unknown”. A small amount of E. coli was obtained using a fired,
sterile inoculating needle. The needle containing the E. coli was used to inoculate the SIM agar
tube labeled “E. coli” by stabbing the semisolid agar with the needle. Next, a small amount of C.
freundii was obtained using a fired, sterile inoculating needle. The needle containing the C.
freundii was used to inoculate the SIM agar tube labeled “C. freundii” by stabbing the semisolid
agar with the needle. Lastly, a small amount of the bacterial unknown as obtained using a fired,
sterile inoculating needle. The needle containing the unknown was used to inoculate the SIM
agar tube labeled “unknown” by stabbing the semisolid agar with the needle. Once all three of
the SIM agar tubes were inoculated, the tubes were incubated for 24 to 48 hours at 37C. After the
tubes were incubated, Kovac’s reagent was added to detect the presence of indole. The tubes
were observed for instances of motility, indole production, as well as sulfide production. 
Methyl Red Assay: Three tubes containing MV-RP broth were obtained. One of the tubes
was labeled “unknown”. Another tube was labeled “E. coli”. The last tube was labeled
“Enterobacter aerogenes”. A small amount of bacterial unknown was obtained using a fired,
sterile inoculating loop. The loop containing the bacterial unknown was used to inoculate the
MV-RP broth tube labeled “unknown”. Next, using a fired, sterile inoculating loop, a small
amount of E. coli was obtained and inoculated into the MV-RP broth tube labeled “E. coli”. Last,
using a fired, sterile inoculating loop, a small amount of E. aerogenes was obtained and
inoculated into the MV-RP broth tube labeled “E. aerogenes”. The tubes inoculated with the
bacteria were incubated for 24 to 48 hours at 37C. After the tubes were incubated, five drops of
methyl red were added to the tubes. The methyl red was dropped in the tubes and allowed to
slide down the side into the broth. The tubes were observed for a color change of red when the
methyl red was added. 
Citrate Assay: Three tubes containing Simmons citrate agar were obtained. One of the
tubes was labeled “unknown”. Another tube was labeled “E. coli”. The last tube was labeled “E.
aerogenes”. A small amount of bacterial unknown was obtained using a fired, sterile inoculating
loop. The loop containing the bacterial unknown was used to inoculate the Simmons citrate agar
tube labeled as “unknown”. The inoculating loop was brushed from side to side on the agar.
Next, using a fired, sterile inoculating loop, a small amount of E. coli was obtained and
inoculated onto the Simmons citrate agar tube labeled “E. coli”. Last, using a fired, sterile
inoculating loop, a small amount of E. aerogenes was obtained and inoculated onto the Simmons
citrate agar tube labeled “E. aerogenes”. The tubes were incubated for 24 to 48 hours at 37C.
After the tube was incubated, the tubes were observed for a color change from green to blue. 
Fat (Triglyceride) Hydrolysis Assay: A plate containing spirit blue agar was obtained and
divided into four quadrants. The quadrants were labeled as follows: “unknown”, “E. coli”, “S.
epidermidis”, and “control”. A small amount of bacterial unknown was obtained using a fired,
sterile inoculating loop and streaked in a single line in the quadrant labeled “unknown”. Next,
using a fired, sterile inoculating loop, a small amount of E. coli was obtained and streaked onto
the quadrant labeled “E. coli” as a single streak. Next, using a fired, sterile inoculating loop, a
small amount of S. epidermidis was obtained and streaked onto the quadrant labeled “S.
epidermidis” as a single streak. The plate was inverted and incubated for 24 to 48 hours at 37C.
After the plate was incubated, the plate was observed for a color change. 
Casein Hydrolysis Assay: A plate of skim milk agar was poured and let to cool. After
cooling, the plate was divided into four quadrants. The quadrants were labeled as follows:
“unknown”, “E. coli”, “Bacillus subtilis”, and “control”. A small amount of bacterial unknown
was obtained using a fired, sterile inoculating loop and streaked in a single line in the quadrant
labeled as “unknown”. Next, using a fired, sterile inoculating loop, a small amount of E. coli was
obtained and streaked onto the quadrant labeled “E. coli” as a single streak. Next, using a fired,
sterile inoculating loop, a small amount of B. subtilis was obtained and streaked onto the
quadrant labeled “B. subtilis” as a single streak. The plate was inverted and incubated for 24 to
48 hours at 37C. After the plate was incubated, the plate was observed for digestion of the casein
in the skim milk agar.  

Results: 

Figure 1: S. marcescens, crystal violet and safranin stain

Figure 2: S. marcescens, nutrient agar

  

Figure 3: S. marcescens (temperature preference, 25C on left and 37C on right), nutrient agar  

Figure 4: S. marcescens, Thioglycolate media 

Figure 5: S. marcescens (Phenol Red Glucose), E. coli (positive control), M.luteus (negative

control), unknown (S. marcescens)

Figure 6: S. marcescens (Phenol Red Lactose), E. coli (positive control), M.luteus (negative

control), unknown (S. marcescens)

Figure 7: S. marcescens (Phenol Red Sucrose), E. coli (positive control), M.luteus (negative

control), unknown (S. marcescens)

 
Figure 8: S. marcescens and E. coli (positive control) (Catalase), nutrient agar  

Figure 9: S. marcescens (SIM Media), E. coli (positive control for presence of indole with

addition of Kovac’s reagent added), C. freundii (positive control for sulfide production),

unknown (S. marcescens) 

Figure 10: S. marcescens (methyl red / MV-RP broth), E. coli (positive control for high conc. of

organic acid production), C. freundii (negative control for high conc. of organic acid production) 

Figure 11: S. marcescens (Bromothymol blue indicator / Citrate), E. coli (positive control for

citric acid uptake / citrate permease), C. freundii (negative control for citric acid uptake / citrate

permease), unknown (S. marcescens)

Figure 12: S. marcescens (Fat (Triglyceride) Hydrolysis / spirit blue agar), E. coli (negative

control for fat hydrolysis), S. epidermidis (positive control for fat hydrolysis), unknown (S.

marcescens)

Figure 13: S. marcescens (casein hydrolysis / casein agar), E. coli (negative control for casein
hydrolysis), B. subtilis (positive control for casein hydrolysis), unknown (S. marcescens)

Table 1: Results from Structural and Culture Characteristic Identification Assays 

Figure Number  Assay Performed  Result 

1 Gram-stain (Gram-positive or Gram-negative)  Gram-negative 

1 Gram-stain (Cell morphology)  Rod 

1 Gram-stain (Cell arrangement) Single 

N/A Endospore  N/A

2 Streak Plate (Pigmentation)  Positive 

 3  Temperature Preference  25C 

4  Oxygen Requirement (Thioglycolate) Facultative anaerobe 

5 Phenol Red Glucose Fermentation Positive (acid production)

6 Phenol Red Lactose Fermentation Negative 

7  Phenol red Sucrose Fermentation Positive (acid production) 

8 Catalase  Positive 

9 SIM Medium (sulfide production) Negative

9 SIM Medium (indole production) Negative 

9 SIM Medium (motility) Positive 

10 Methyl Red  Negative 

11 Citrate  Positive 

12 Fat Hydrolysis Negative 

13 Casein Hydrolysis  Negative 

To identify a bacterial unknown, a Gram-stain was applied to the unknown. Results

indicate that the unknown bacteria was Gram-negative. This led to the endospore stain test not
being completed. The bacterial unknown was incubated at two temperatures, 25C and 37C, to
see the prefered temperature at which the bacteria will grow. The results indicated that the
bacterial unknown grew more efficiently at 25C. A thioglycolate media was inoculated with the
bacterial unknown to test for oxygen requirement. It was determined the bacteria was an
facultative anaerobe. The bacterial unknown was inoculated into three carbohydrate fermentation
assay tubes with glucose, lactose, and sucrose. These tests looked for the ability of the bacteria to
ferment specific sugars (glucose, lactose, and sucrose) and the production of CO2 gas indicated
by a bubble in the durham tube. The results indicated no fermentation or CO2 production from
lactose but fermentation of glucose and sucrose without CO2 production in both tests. A catalase
assay was performed to detect the presence of catalase enzyme and results indicated the bacterial
unknown had catalase enzyme. SIM agar was used to determine sulfide production, indole
production and if motility was evident. The SIM assay resulted in a negative result for sulfide
production as well as indole when the Kovac’s reagent was added after incubation occured.
However, the SIM assay resulted in a positive result for motility. Using MV-RP broth to detect
the quantity of acid production by fermentation, a methyl red assay resulted in a negative result
for high concentration production of acid. A citrate assay was performed to look for citrate as the
sole carbon source, the test resulted in a positive result of citrate as a sole carbon source. A fat
hydrolysis assay was used to look for lipases. The results indicated absence of lipase present
resulting in no lipid digestion. A casein hydrolysis test was performed to indicate the presence of
proteases and results indicated a negative result of skim milk protein casein hydrolysis. 

Discussion: 
The identification of a bacterial unknown in a patient suffering from a debilitating illness
is crucial for their survival in terms of identifying treatment options and the best chance to have
minimal complications after treatment has concluded. Also, the identification of a bacterial
unknown needs to be made in a timely manner as excess time may decrease the chance of
survival. Through a variety of structural and biochemical tests, the identification of a bacterial
unknown was found. Structural and biochemical characteristic results interpreted together give
the microbiologist the best chance of correctly identifying a bacterial unknown because if only
one type of characteristic identified, such as structural or more specifically Gram staining results,
may not be sufficient enough to make a definitive conclusion (Pomerville 2018). Through the
identification process of structural and biochemical characteristics, unknown 16 as provided by
the instructor was correctly identified as S. marcescens. The bacteria S. marcescens is of great
interest as the bacteria is an opportunistic pathogen (Pomerville 2018). S. marcescens is a
common cause of nosocomial infections (Kim 2020). The bacteria is known to cause many
infectious diseases such as urinary and respiratory infections as well as wound infections and
life-threatening bacteremia (Kim 2020). As time goes on, just as many other bacteria, S.
marcescens is becoming a multidrug-resistant (MDR) bacteria that causes outbreaks that produce
a mortality rate of 25% - 58% (Kim 2020). Identifying S. marcescens would allow the caretaker
of the infected patient to allocate treatment options for the best chance of survival. The Gram-
stain technique, which was the first assay completed when identifying the bacterial unknown and
probably the most important staining procedure in microbiology, determined that S. marcescens
was a Gram-negative bacteria (Pomerville 2018). This assay differentiates the great majority of
bacteria into two major categories, Gram-positive or Gram-negative (Pomerville 2018). Each of
these bacteria due to their Gram identification will guide the treatment options as Gram-positive
and Gram-negative bacteria contain different virulence factors that can be targeted by antibiotics,
such as the cell wall components (Foster 2018). Also, due to the result of S. marcescens being a
Gram-negative bacteria, an endospore stain test was not needed because generally, a gram-
negative bacteria does not produce endospores. Not performing the test saves time in discovering
the identity that can be crucial in a medical emergency. Next, cell morphology and cell
arrangement was also viewed under a microscope and was determined to be a rod shaped
bacteria and appeared as a single bacteria. By identifying the morphology and cell arrangement
of the bacteria further narrows down the possibilities because different bacteria are characterized
by different cell shapes and arrangements (Pomerville 2018). Next, a temperature preference
assay is used to determine the optimal temperature in which S. marcescens grows. The assay
concludes that the bacteria grew preferably at 25C rather than 37C. This is significant due to the
fact that microbes cannot regulate their temperatures like humans can, so the microbe has to be
in an environment where it can grow effectively (Foster 2018). Thus, knowing the optimal
temperature in which the S. marcescens or other bacteria for that matter can guide the treatment
or prevention. Changes in temperature can affect the bacteria in many ways such as genomic
stability, membrane fluidity, and enzyme function. If these components of the bacteria are
destroyed or hindered in ways such as raising temperatures like cooking food, the bacteria can be
destroyed (Foster 2018). Oxygen requirements of S. marcescens is tested via thioglycolate
media. The media shows the growth of the bacteria in various levels of the tube. The media is a
differential media that will distinguish between aerobes and anaerobes by the requirement of
oxygen. The growth of S. marcescens was noted as a higher concentration of growth near the
surface of the agar with diffusion to the base of the tube. Thus, the determination of facultative
anaerobes was concluded. The significance of the identification of S. marcescens being a
facultative anaerobe means that the bacteria possess components for both fermentation as well as
aerobic respiration besides the tools for anaerobic respiration. So, depending on the oxygen
availability, the bacteria can grow in a wide variety of environments (Foster 2018). Also, the
growth of the bacteria in a wide variety of environmental conditions will support the findings of
S. marcescens in multiple environments. The above characteristics were cultural and structural
characteristics. Alone, these are not sufficient in identifying a bacterial unknown with almost
certainty (Pomerville 2018). So, biochemical characteristics such as the presence or absence of a
substrate or product and enzyme identification can further confirm an unknown bacteria
(Pomerville 2018). Fermentation of various carbohydrate sources, in the case S. marcescens,
glucose and sucrose was fermented. Also, it is important to note that the other carbohydrate
source, lactose, was not fermented. The results from this assay are significant in that S.
marcescens ferments glucose and sucrose but not lactose. Thus, comparing these findings to
other bacteria that are known to ferment different combinations or just single carbohydrate
sources distinguishes S. marcescens from other bacteria. Also, with the fermentation of
carbohydrate sources, the bacteria can also produce carbon dioxide gas. However, S. marcescens
does not produce carbon dioxide in any of its fermentation processes. Again, the importance of
the lack of carbon dioxide formation is used to distinguish S. marcescens from another bacteria
that may produce carbon dioxide through fermentation processes. Next, continuing with the
different assays to narrow down the identification of an unknown bacteria, a catalase assay is
performed. The assay is the first one in this experiment that looks for the presence of an enzyme.
The enzyme identified in this assay is the catalase enzyme which is an enzyme that breaks down
hydrogen peroxide to oxygen and water (Pomerville 2018). Effervesce was evident due to the
fact that S. marcescens contains the catalase enzyme. This again will help distinguish S.
marcescens from other bacteria. Another test, using SIM agar, will detect the production of
sulfide indicated by hydrogen sulfide gas. Also, this assay will detect the presence of indole.
Indole production indicates the presence of an enzyme called tryptophanase which breaks down
tryptophan (Reay 2020). The indication of both the production of hydrogen sulfide and indole
was negative in S. marcescens which follows what was known of the bacteria, further narrowing
down the possibilities. The “M” in the SIM agar assay looks for motility. Motility will occur
when a bacteria possesses some sort of appendage, like a flagella, to swim. The SIM agar
appeared cloudy, indicating the unknown bacteria has an appendage that enables the bacteria to
move (Reay 2020). So, this was also indicative that S. marcescens was a possible contender for
the identification as it is known it contains flagella. Flagella increase the virulence of S.
marcescens and this probably aids the bacteria in producing the high mortality and outbreaks that
are becoming apparent in the past few decades (Haiko 2013). Another test, relating to the
fermentation of glucose which is apparent in S. marcescens is the methyl red test. The methyl red
test indicates the presence of a high concentration of organic acids produced by fermentation of
glucose (Reay 2020). The presence of this high concentration was expected but not definite.
Again, this test just adds to the verification of S. marcescens as the unknown bacteria. Simmons
citrate agar was used as well to determine the identity of the bacterial unknown. It was suspected
and found that S. marcescens did react positively to the assay. Just like in the SIM and catalase
assays, the Simmons citrate agar test is looking for the presence of an enzyme known as citrate
permease (Reay 2020). The enzyme indicated that the bacteria uses citric acid as its sole energy
source by the uptake of citrate into the cells (Reay 2020). Keeping with the enzyme identification
processes, a fat (triglyceride) hydrolysis is completed on an unknown bacteria to look for lipase
enzymes (Pomerville 2018). These enzymes break down triglycerides into glycerol and fatty acid
molecules (Pomerville 2018). It is suspected that S. marcescens would not contain lipase
enzymes and not hydrolyzed triglyceride. This was confirmed by a negative test indicating no
hydrolysis occurs on the spirit blue agar. Rounding out the structural and cultural characteristic
assays, especially the enzyme identification assays, is the casein hydrolysis assay. The assay tests
for the hydrolysis of protein, specifically in skim milk agar, to look for proteinases (Pomerville
2018). The assay specifically looks for the proteinase called caseinase. It is suspected that S.
marcescens would not contain such enzyme and this is confirmed in a negative result from the
casein hydrolysis assay. There were not any significant or notable problems that occurred during
the experimental parts of the bacterial identification process. All of the tests above provide
results that are used to narrow down and eventually pointed to a possible identity of a bacterial
unknown. The results obtained from the different tests are compared to charts and literature that
contain documentation of the characteristics. Careful evaluation of the results will hopefully
match the unknown bacteria to one of known origin. There needs to be little error, such as
contamination, that may upset the results leading to a misdiagnosis of a bacteria. Misdiagnosis of
an unknown bacteria can result in treatment options, such as antibiotics or supportive therapies,
to have adverse reactions or be too limited to help combat the bacteria (Kim 2015). S.
marcescens was correctly identified by using assays to determine cultural and biochemical
characteristics as well as using knowledge from negative tests to guide the experiment forward.
S. marcescens was compared to “fingerprint” characteristics in the chart to distinguish it from
other bacteria in the environment. 

References 
Foster J W., Aliabadi Z, Slonczewski J L. 2018. Microbiology: The Human Experience. New
York (NY) 
Haiko, J., & Westerlund-Wikström, B. 2013. The Role of the Bacterial Flagellum in Adhesion 
and Virulence. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009794/ 
 Kim, S. B., Jeon, Y. D., Kim, J. H. 2015.. Risk factors for mortality in patients with Serratia 
marcescens bacteremia. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329343/ 
Pommerville JC. 2018. Laboratory Fundamentals of Microbiology. Eleventh Edition. Burlington
(MA) 
Reay, J. 2020. Exercise 60 and 61 BL 219 Microbiology Lab. Retrieved from
https://drive.google.com/file/d/1s3HTa8_lkomyhLrcSi54X7E6jgaJQRO7/view