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The Examination and Identification of a Bacterial Unknown

Abby Herman, Bailey Julian, and Lauren Hennessy

SBL 219 03

13 May 2021

Abstract:

A bacterial unknown was obtained with the main objective of identifying the bacterial

unknown through various methods. Bacterial structural, functional characteristics, and

biochemical tests including but not limited to carbohydrate fermentation, catalase production,

triglyceride and casein hydrolysis were conducted to identify the bacterial unknown. After all the

tests were conducted the bacterial unknown was determined to be Enterobacter aerogenes.

Introduction:

The identification of a bacterial unknown is an integral part of Microbiology, as it is

useful in multiple areas such as clinical diagnoses, public health, monitoring food safety,

environmental monitoring, and in the identification of biological threat agents (Emerson 2008).

Without the ability to study and identify an unknown bacteria, infection, water and food

contamination, and a lack of antibiotic study would result. The need for bacterial identification

dates back to their discovery by Antoni van Leeuwenhoek in the 1670’s (Tsang 2020). Thereafter

the characterization and identification of newfound and known bacteria were paramount in the

progression of microbiology and the safety and medical treatment of human beings. The way

microbes were initially characterized was purely on size, shape, and the dyes absorbed (Tsang

2020). The techniques evolved and became more refined, allowing for more bacteria to be

further categorized. Various types of agar were created, facilitating growth for bacteria to be

studied and described. This included selective and differential media. Selective media both
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inhibits bacteria and helps facilitate the growth of a specific bacterium (Tsang 2020). Differential

media highlights the differing growth patterns exhibited by multiple bacterial species (Tsang

2020). Biochemical tests and staining are also used to identify bacteria and their characteristics.

For example, some biochemical tests see if particular enzymes are present in the organism.

While microscopy stains can highlight structure of the bacteria like a Gram Stain. Additional

tests and characteristics that aid in narrowing down a bacterium include: Temperature preference,

Motility, Endospore stain, Growth form on agar, Growth in broth, Pigmentation, Oxygen

requirement, Sugar fermentation, Indole production, Starch hydrolysis, Casein Hydrolysis, H2S

gas production, etc (Pomerville 2017). Using these tests as well as additional ones, a study to

identify characteristics of a bacterial unknown was performed to determine the species of

bacteria based off of the possible unknowns in Table 1.

Methods:

Bacterial Structural Characteristics:

A broth culture of an unknown bacterium identified by only a letter, number, or both was
obtained. An air-dried, heat-fixed smear from the broth culture that was created and used
in a gram stain. The shape, relative size, cell arrangement, and its reaction to the stain
were then determined. Determine if the unknown produces endospores. If so, complete an
endospore stain.

Temperature Preference:
Two nutrient agar slants were created. Each slaent was inoculated with the unknown
bacterium. One slant was incubated at 37℃ for 48 hours, while the other one was at room
temperature. The better growth temperature of the unknown was determined. Keep One
slant was kepts as a stock culture for further experiments. Results were then recorded.

Colony Appearance on Agar:

One Nutrient agar plate was prepared. A sharpie was used to label the plate with names
and the date. A section of the plate was inoculated with a loopful of bacteria, and a streak
plate was then created. The plate was then inverted and incubated. The plate was
observed for the quantity and quality of growth of the unknown bacteria. Results were
then recorded.

Appearance in Nutrient Broth:

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A tube of sterile nutrient broth was obtained, then labeled with names, the date, and the
temperature that it was incubated at. The tube was inoculated with the unknown bacteria.
The tube was then incubated at the designated temperature for the indicated amount of
time, and then refrigerated until it was time to observe. The tube was then examined, and
the characteristics of the broth were recorded.

Oxygen Gas Requirement:

A thioglycollate medium tube was obtained and labeled with names, the date, and the
temperature that it was incubated at. The tube was then inoculated with a loopful of the
unknown bacteria. The tube was swirled and gently rotated to mix the bacteria. It was
then incubated at room temperature for 24 to 48 hours. The tube was examined and the
location of growth was recorded.

Biochemical Characteristics of Bacteria: Carbohydrate Fermentation:

Tubes of various carbohydrate media were labeled with names, the date, and the bacteria.
Each tube was inoculated with the loop of bacteria, then placed back into the rack. Each
tube was flamed after removing the cap, the bacteria was placed into the tube, the tube
was reframed, then the cap was put back on before it went into the rack. This process was
repeated for every tube. The tubes were incubated for 24-48 hours at 37 degrees celsius,
and a set of uninoculated tubes were set to ba a control. Each tube was observed for a
color change. Results were then recorded.

Catalase Production:
A nutrient agar plate was obtained and labeled on the bottom side with names, the date,
the medium name, and bacteria name. After hardening, the plate was divided into halves.
One half was inoculated with Escherichia coli and the other half was inoculate with the
unknown bacteria. The plate was incubated invertedly for 24-48 hours at 37 degrees
celsius. Drops of 3% hydrogen peroxide were directly placed onto the bacterial growth to
test for the presence of catalase. Results were then recorded.

Hydrogen Sulfide Production:

A tube of SIM medium was obtained and labeled with names, the date, and the
temperature that it was incubated at. An inoculating needle was used to pick up bacteria
and stab into the semi solid agar about 3/4th the way down. The needle was withdrawn
the same way it went in. The tube was inoculated for 24-48 hours at 37 degrees celsius,
then refrigerated along with a positive and negative control. The tubes were then
observed to identify the production of hydrogen sulfide. Results were then recorded.

IMViC Series:
MR-VP broths and Simmons Citrate agar slants were inoculated with the bacterial
unknown, E.coli, and E.aerogenes. Five drops of methyl red solution were added to each
MR-VP tube. All of the tubes were observed for a color change and bacterial growth.
Results were then recorded.
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Fat (Triglyceride) Hydrolysis:

A plate of blue spirit agar was obtained. After hardening, the plate was inoculated with
S.epidermidis, E.coli, and the bacterial unknown. The plate was inverted and incubated
for 24-48 hours at 37 degrees celsius. The plate was then examined for a dark or light hue
around the streak. Results were then recorded.

Casein Hydrolysis:
A plate of skim milk agar was obtained. After hardening, the plate was inoculated with
the bacterial unknown, E.coli, and B.subtilis. The plate was inverted and incubated for
24-48 hours at 37 degrees celsius. The plate was then examined to see if the area around
the streak was cloudy or clear. Results were then recorded.

Results:

Table 1: Some Characteristics of Selected Bacterial Species

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Figure 2: Streak Plate of unknown at 25℃.

Figure 1: Gram-stain of the bacterial
Although single colonies did not culture, the
unknown indicating a Negative Gram stain
form appears to be circular, the margin
and rod shaped bacteria in single cell
entire, the bacteria is slightly elevated, and
arrangement.
the pigment is tan colored.

Figure 3: To determine temperature Figure 4:To determine temperature

preference a Nutrient Agar Slant of preference a Nutrient Agar Slant of
unknown at 25℃ was performed. There was unknown at 37℃ was performed. There was
bacterial growth, this demonstrates the bacterial growth, this demonstrates the
bacteria can survive and thrive at 25℃. bacteria can survive and thrive at 37℃.

Figure 5: Nutrient Broth with unknown
displaying no growth at room temperature
(25℃).
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Figure 6:
Thioglycollate Medium with unknown
displaying turbid growth throughout and a
pellicle at the top of the tube, indicating
facultative aerobic growth.
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Table 2: Results of the Carbohydrate Fermentation Test

The unknown was able to ferment Lactose, Glucose, and Sucrose as well as produce CO2 and
acid as indicated by the gas in the Durham tube and the yellow colored Phenol Red media.
Phenol Red- Lactose Phenol Red-Glucose Phenol Red- Sucrose

Unknown Acid and Gas Produced Acid and Gas Produced Acid and Gas Produced

E. coli Acid and Gas Produced Acid and Gas Produced No Acid or Gas
Produced

M. luteus Acid and Gas Produced No Acid or Gas No Acid or Gas

Produced Produced
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Figure 7: Catalase Production Test of unknown and E.coli was positive in both bacteria,
indicating that the Catalase enzyme is produced in both species and they can break down
Hydrogen Peroxide.

Figure 8: SIM Agar Inoculated with C. Figure 9: SIM Agar Inoculated with
freundii indicating motility throughout the unknown showing slight motility and no
agar and the enzyme cysteine desulfurase production of Hydrogen Sulfide.
producing Hydrogen Sulfide.
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Figure 10: SIM Agar Inoculated with E. coli

showing slight motility and no production of Hydrogen Sulfide.

Figure 11: MRVP of E. aerogenes indicates Figure 12: MRVP of unknown indicates it is
it is not able to ferment glucose and acid not able to ferment glucose and acid was not
was not produced. produced.
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Figure 13: MRVP of E.coli indicates it is Figure 14: Citrate Assay of unknown
able to ferment glucose and acid was indicates that it can use citrate as a sole
produced. carbon energy source for growth by the blue
color.

Figure 15: Citrate Assay of E. aerogenes Figure 16: Citrate Assay of E. coli
indicates that it can use citrate as a sole indicates that it cannot use citrate as a sole
carbon energy source for growth by the blue carbon energy source for growth by the blue
color. color.
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Figure 17: Spirit Blue Agar Plate with
Staphylococcus epidermidis, E. coli, and
unknown. The S.epidermidis was positive
and the blue color indicated that lipid
digestion had occurred. The unknown and E.
coli did not digest the lipids according to
their lighter color.
Figure 18: Skim Milk Agar Plate with
Bacterial unknown, E. coli, and Bacillus
subtilis. B. subtilis digested the casein
because of the clear agar next to the streak.
However the unknown and E. coli did not as
seen by their cloudy appearance.

Discussion:

To begin the progress of identifying the bacterial unknown the bacterial structure was

tested through a gram-stain. The gram-stain was interpreted to be gram-negative species which

limited the bacterial unknown options to Eschericia coli, Alcaligenes faecalis, Citrobacter

freundii, E. aerogenes, Pseudomonas fluorescens, Neisseria subflava, and Serratia marcescens

(figure 1). The bacterial culture characteristics were determined to have a temperature preference

of 37℃ and when plated on nutrient agar was tan rod-shaped, single cell arrangement (figure 2, 3

& 4). These characteristics limited the bacterial unknown further to E.coli, A. faecalis, C.

freundii, E. aerogenes, and P. fluorescens.

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The first biochemical test conducted was the carbohydrate fermentation. Carbohydrate

fermentations test the bacteria’s ability to ferment a sugar into an acid or an acid and gase. The

specific sugars that were glucose, sucrose, and lactose. The 1 phenol-red glucose for the

unknown was determined to be an acid and gas producing due to the gas bubble seen in durham

tube and the yellow color which can be seen in table 2. The 1 phenol-red glucose for E. coli was

determined to be an acid and gas producing due to the gas bubble seen in durham tube and the

yellow color which can be seen in table 2. The 1 phenol-red glucose for M. luteus was

determined to be negative for acid and gas production due to the absence of a gas bubble in

durham tube and the red color which can be seen in table 2. The 1 phenol-red sucrose for the

unknown was determined to be an acid and gas producing due to the gas bubble seen in durham

tube and the yellow color which can be seen in table 2. The 1 phenol-red sucrose for E. coli was

determined to be negative for acid and gas production due to the absence of a gas bubble in

durham tube and the red color which can be seen in table 2. The 1 phenol-red sucrose for M.

luteus was determined to be negative for acid and gas production due to the absence of a gas

bubble in durham tube and the red color which can be seen in table 2. The 1 phenol-red lactose

for the unknown was determined to be an acid and gas producing due to the gas bubble seen in

durham tube and the yellow color which can be seen in table 2. The 1 phenol-red lactose M.

luteus was determined to be an acid and gas producing due to the gas bubble seen in durham tube

and the yellow color which can be seen in table 2. The 1 phenol-red lactose E. coli was

determined to be an acid and gas producing due to the gas bubble seen in durham tube and the

yellow color which can be seen in table 2. From these results the bacterial unknown species was

further narrowed down to C. freundii and E. aerogenes. The next biochemical test performed was

catalase production which tests if the bacteria is a catalase producer. The E.coli and unknown
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bacteria both tested positive for being a catalase producer which was indicated by the formation

of bubbles when the hydrogen peroxide was added as seen in figure 7. Keeping in mind the

results of the prior test the two bacterial species thought to be the unknown were C. freundii and

E. aerogenes.

Hydrogen sulfide production was the next biochemical test conducted and looked for the

ability of the organism to produce hydrogen sulfide and the motility of the organism. A positive

test for SIM agar is seen by the darkening or black color of the agar, while a negative test

remains the initial tan color. Motility is concluded through whether the agar is cloudy or clear.

Cloudiness throughout the agar is a positive test for the motility of the bacteria. The unknown

bacteria was positive for motility and hydrogen sulfide production as seen by the cloudiness and

darkness in figure 9. E. coli tested negative for hydrogen sulfide which was concluded from the

tan appearance of the agar; however, cloudiness was seen in the agar (figure 10). C. freundii was

positive for both hydrogen sulfide production and motility as seen in figure 8. These results

were unable to help with the narrowing down of the prior two bacterial species thought to be the

unknown: C. freundii and E. aerogenes.

IMViC series is a series of tests however for the identification of the bacterial unknown

only methyl red assay and a citrate assay was conducted. Methyl red assay distinguishes the

quantity of acids produced by fermentation of the bacteria. A positive test for high concentration

of acids produced by fermentation results in an orange to red color after the addition of the

methyl red. A negative reaction for high concentration of acids by fermentation results in the

medium being yellow after the addition of methyl red. E.coli showed a positive test result

meaning it produces large amounts of glucose as seen in figure 13. E. aerogenes showed a

negative test result meaning that it does not produce large amounts of glucose as seen in figure
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11. The bacterial unknown is also negative meaning that it does not produce large amounts of

glucose as seen in figure 12. The citrate assay detects the ability of bacteria to use citrate as its

sole source of energy. As seen in figure 16, E. coli was negative meaning that it is unable to use

citrate as its sole source of energy. E. aerogenes was positive meaning that it is able to use citrate

as a source of energy and turned blue indicating so as seen in figure 15. The bacterial unknown

was positive meaning that it is able to use citrate as a source of energy and turned blue indicating

so as seen in figure 14. These results were unable to help with the narrowing down of the prior

two bacterial species thought to be the unknown: C. freundii and E. aerogenes.

One of the last biochemical tests conducted was fat (triglycerides) hydrolysis which is

done on spirit blue agar. The spirit blue agar is rich in triglycerides and therefore can test the

bacteria’s ability to digest triglycerides and in turn the pH changes. If a positive reaction for fat

hydrolysis occurs the bacteria can digest lipids and in turn the pH is lowered which is shown by

the deep blue appearance of the agar. A negative reaction cannot digest lipids and the pH remains

the same which is apparent by a lavender color of the agar. S. epidermidis is positive for lipid

digestion which is why there are portions of dark blue seen in the middle of the bacterial streak

(figure 17). E.coli is negative for lipid digestion which is why there is no blue portion where the

streak took place and the agar appears lavender (figure 17). The unknown bacteria was also

negative for lipid digestion, which was confirmed by comparing the unknown bacteria to the

E.coli (figure 17). The last biochemical test conducted was casein hydrolysis which tests for the

ability of cells to produce proteases. A positive casein hydrolysis test will cause the skim milk

agar the medium will become clear around cells because of the casein enzyme. A negative result

is that the medium remains opaque because casein is not produced. The positive control for this

experiment was the B. subtilis as digestion has occurred throughout the streak (figure 18). The
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negative control for the experiment was E.coli as it remained cloudy throughout the streak

(figure 18). The unknown was determined to be negative as cloudiness on the skim milk agar

remained as seen in figure 18. Through fat and casein hydrolysis it was concluded that the

bacterial unknown was E. aerogenes. The organism was identified correctly through the

confirmation of the professor.

E. aerogenes is known to be opportunistic and a multi resistant bacterial pathogen during

the past three decades and has affected many hospital wards (Regli & Pagès, 2015). It is also

known to be a species in which there is the ability of genetic shift increasing their antibiotic

resistance. This allows for E. aerogenes to rapidly colonize their host and adapt their metabolism

to their external environment. The adaptability was seen in the laboratory as E. aerogenes grew

at 37℃ and 25℃.

References:

Emerson, D., Agulto, L., Liu, H., & Liu, L. (2008). Identifying and characterizing bacteria in an

era of genomics and proteomics. Bioscience, 58(10), 925–936.

https://doi.org/10.1641/B581006

Davin-Regli, A., & Pagès, J.-M. (2015). Enterobacter aerogenes and Enterobacter cloacae;

versatile bacterial pathogens confronting antibiotic treatment. Frontiers in Microbiology,

6. doi:10.3389/fmicb.2015.00392

Pommerville, J. C. (2017). Laboratory Fundamentals of Microbiology (11th ed.). Sudbury, MA:

Jones and Bartlett.

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Tsang, J. (2020, February 7). Identifying bacteria through look, growth, stain and strain.

Retrieved May 13, 2021, from Asm.org website:

https://asm.org/Articles/2020/February/Identifying-Bacteria-Through-Look,-Growth,-Stai