Shereen Richard

Enterobacter aerogenes
BIOL 2325 Section 3
7/22/14

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Introductory Paragraph:
During this semester in BIOL 2325 Section 3, 11 labs were performed. This course contained
labs that included using aseptic technique, cultivating, streaking, and isolating different types of bacteria on
different types of media, using different staining techniques to identify microbial organisms, using a
microscope properly in order to identify organisms effectively, using selective and differential media to
identify properties of different microorganisms, performing enzymatic tests to help in the identification of
microbes, and using many other techniques to help identify certain bacteria and microbes. All of these
techniques are very helpful in identifying microbial organisms. For this report and experiment, these
techniques were applied to help in the identification of an unknown organism, #17, provided by Ms.
Simms. Unknown Organism #17 was provided as a pure culture in a broth media. To begin the
identification process, a sample of the broth was taken and, using the streaking for isolation technique, the
bacteria were isolated on a nutrient agar plate. Also, a sample of the broth was stained using the Gram Stain
technique to begin the identification of Unknown Organism #17s morphology, arrangement, size, and cell
wall structure. After these procedures were completed, the unknown sample was streaked on other media
such as Eosin Methylene Blue agar to help in the identification and confirmation of the unknown
organisms cell wall structure and lactose fermentation properties. After obtaining the information from
these procedures, other procedures and tests were performed to effectively identify Unknown Organism
#17. To help with the process of identification, Ms. Simms provided a flowchart to help in the identification
of the unknown organism based on its morphology, arrangement, cell wall structure, fermenting abilities,
and other enzymatic properties. Ms. Simms and Ms. Voth were also there throughout the semester to help in
the identification process.
Materials/Methods:
*All of these procedures are retrieved from the procedures provided on Moodle from Professor
Simms (Procedures 2, 3, 4, 6, 8, 11).
A. Streaking for Isolation Procedure (Procedure 2: (Leboufe & Pierce, 2013):
*Label all agar plates, test tubes, and slides with date, class and section, initials, type of medium
or stain being used, and name of sample.
Materials/Equipment: Inoculating loops, Bacti-cinerator, agar plate (labeled with quadrants), broth
containing bacteria, incubator, safety equipment (Procedure 2: Leboufe & Pierce, 2013).

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An inoculating loop was obtained and sterilized. After letting the loop cool, a loop full of broth was
obtained from the broth culture. After dividing the agar plate into four quadrants, Quadrant #1 was
streaked carefully with the loop. The loop was re-sterilized and cooled. Quadrant #2 was streaked and
included 1-2 streaks from Quadrant #1. After this step, the previous quadrant was not streaked again.
These steps were followed for Quadrant #3 and Quadrant #4. When finished streaking, the loop was
sterilized, and the plates were incubated upside down at 37 degrees Celsius for 24 hours (Procedure 2:
Leboufe & Pierce, 2013).
B. Gram Stain Procedure (Procedure 4: Leboufe & Pierce, 2013 ):
*Label all agar plates, test tubes, and slides with date, class and section, initials, type of medium
or stain being used and name of sample.
A glass slide was obtained, and a smear was prepared using aseptic technique. The smear was prepared
by applying a loop full of the bacterial broth with a sterile inoculating loop to a clean slide and
spreading the cells to the size of a dime. The slide was allowed to dry and was heat fixed over a hot
plate to obtain the smear. After the smear was obtained, the slide was placed on a staining rack, was
flooded with crystal violet, was left to sit for one minute, and was then rinsed with deionized water.
The second stain applied was Grams Iodine. The stain was left to sit for one minute and then was
rinsed with deionized water. The slide was then treated at a 45-degree angle with 10 drops of 95
percent ethanol and was immediately rinsed with deionized water. The slide was then flooded with
Safranin, was left to sit for one minute, and then was rinsed with deionized water. The slide was then
blotted with bibulous paper and was viewed under the oil immersion lens of the microscope (Procedure
4: Leboufe & Pierce, 2013).
Procedure for using the 100x (oil immersion) objective lens (Procedure 3: Leboufe & Pierce,
2013):
Because the microscope is par focal, the specimen was first focused on the 10x objective and then
the 40x objective. The objective lenses were placed in the middle of the 40x and the oil immersion
lens, and a drop of oil was placed on the slide. The slide was returned to the stage and was focused
with the oil immersion lens. After viewing, all lenses were wiped with lens paper (Procedure 3:
Leboufe & Pierce, 2013).
C. Selective and Differential Media (Eosin Methylene Blue agar) (Procedure 6: Leboufe & Pierce,
2013 ):

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*Label all agar plates, test tubes, and slides with date, class and section, initials, type of medium
or stain being used, and name of sample.
Materials: Bacti-cinerator, inoculating loop, culture, Eosin Methylene Blue agar (Procedre 6: Leboufe
& Pierce, 2013).
Procedure:
A sample of bacteria was obtained and was streaked onto the Eosin Methylene Blue agar using aseptic
technique. The agar was incubated at 37 degrees Celsius overnight and then refrigerated (Procedure 6:
Leboufe & Pierce, 2013).
D. H. Oxidase Test Procedure (Procedure 8: Leboufe & Pierce, 2013):
*Label all agar plates, test tubes, and slides with date, class and section, initials, type of medium
or stain being used, and name of sample.
Two drops of oxidase reagent were placed onto a piece of filter paper. A bacterial colony was obtained
on a wooden applicator stick and was smeared onto the site with the oxidase reagent on the filter paper.
A color change was looked for (Procedure 8: Leboufe & Pierce, 2013).
E. Citrate Test Procedure (Procedure 8: Leboufe & Pierce, 2013):
*Label all agar plates, test tubes, and slides with date, class and section, initials, type of medium
or stain being used, and name of sample.
Two citrate slants were obtained. One slant was streaked with a sample of bacteria, and the other slant
was used as a control. Both slants were incubated at 37 degrees Celsius for 24 hours. A color change in
the medium was looked for (Procedure 8: Leboufe & Pierce, 2013).
F. Sulfur, Indole, Motility (SIM) Test Procedure (Procedure 8: Leboufe & Pierce, 2013):
*Label all agar plates, test tubes, and slides with date, class and section, initials, type of medium
being used, and name of sample.
A SIM deep was inoculated by stabbing the deep with an inoculating loop containing a sample of
bacteria. The deep was incubated at 37 degrees Celsius for 24 hours. After the incubation period ended,
Kovacs reagent was added. For indole production, a color change was looked for. For sulfur
production, black medium was looked for. For motility, a fuzzy stab was looked for (Procedure 8:
Leboufe & Pierce, 2013).
G. Urease Activity: Identification of Proteus spp. Procedure (Procedure 8: Leboufe & Pierce, 2013):
*Label all agar plates, test tubes, and slides with date, class and section, initials, type of medium
being used, and name of sample.
A urease slant was obtained and was streaked with bacteria using and inoculating loop and aseptic
technique. The slant was incubated at 37 degrees Celsius for 24 hours. For urease activity, a color
change in the medium was looked for (Procedure 8: Leboufe & Pierce, 2013).
H. Microbial Genetics Test Procedure (Procedure 11: Leboufe & Pierce, 2013 ):

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*Label all agar plates, test tubes, and slides with date, class and section, initials, type of medium
being used, and name of sample.
Materials:
Dry ice, Ethanol (alcohol), Distilled water, Hot water bath, Microcentrifuge tubes, Microcentrifuge,
Vortexer, Micropipettes, Micropipette tips (Procedure 11: Leboufe & Pierce, 2013).
Procedure:
PART A
A microcentrifuge tube was obtained, 500 ul of broth culture was placed into it, and was centrifuged
for five minutes at maximum speed. After centrifuging, the supernant was discarded. The remaining
pellet at the bottom of the tube was re-suspended in 500 ul of distilled water and was mixed by the
means of a vortex. The tube was then placed in an ethanol ice bath to freeze for three minutes, and then
was transferred into a hot water bath for three minutes. The ice bath and hot water bath step was
repeated twice with vortexing in between. The mixture was then centrifuged for five minutes on
maximum speed. After the centrifuging was complete, the supernant was transferred into a new
microcentrifuge tube (Procedure 11: Leboufe & Pierce, 2013).

PART B. Polymerase Chain Reaction

Materials:
NH4/MgCl2 PCR Buffer, Taq Polymerase, Forward 16S Primer, Reverse 16S Primer, Purified
Unknown DNA, Micropopettors, Micropipettor tips (Procedure 11: Leboufe & Pierce, 2013).
Procedure
Twenty-two ul of PCR buffer was placed into the PCR tube. Eight ul of each of the forward and
reverse primers, ten ul of purified DNA from Part A, and two ul of Taq polymerase were added to
the PCR tube and vortexed. The samples were left on ice until the PCR reactions were performed
in the thermocycler (Procedure 11: Leboufe & Pierce, 2013).
Results/Charts:
PART A: Non-enzymatic Tests

Figure 1: Gram Stain

Inferences: After performing a Gram Stain
on the broth containing Unknown Organism
#17, the results were similar to Figure 1. The
bacteria were gram-negative bacillus.

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Table 1 (Refer to Figure

Observations of Growth on

2)

Nutrient Agar:

Is the colony pigmented?

Yellow Tint

What color is it?

Is the colony transparent

Transparent

or opaque?
Does the colony have

Smooth Edges

rough or smooth edges?

Is the colony large or

Figure 2: Nutrient Agar Plate (Streaking for

Small/Scattered

Isolation)

small?
Is the colony flat or raised

Inferences: This nutrient agar plate contains the

Flat

results of Unknown Organism #17 after streaking

in the center?
Is the colony dry, creamy,

for isolation. There were a few individual colonies

Moist looking

Inferences:
Nutrient
agar is useful
the
in quadrants
3 and
4, concluding
that theinisolation

or wet looking?
(Rakusin,

characteristics
of the bacterial
was identification
successful. Toof
confirm
that the isolation
was

2014)

colony asuch
as color,
odor, and
successful,
second
Gramsize,
Stainshape,
was performed

Table 1:

Unknown
Organism
#17 colonies
had a
fromtexture.
the isolated
colonies
on the nutrient
agar plate.

Nutrient

tint, were
edges,
The yellow
Gram Stain
from transparent,
the broth andhad
thesmooth
Gram Stain

Agar

small and
scattered,
were flat,
and seemed
fromwere
the nutrient
agar
plate showed
the same
result to
be moist.
confirming
there was a pure sample on the plate
with no contaminations.

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Table 2 (Refer to Figure 1)

Organism
Shape
Arrangement
Color After Staining
Gram Positive or Gram Negative?
Table 2: Gram Stain

Observations of Gram Stain Slide:

Unknown Organism #17
Bacillus
Bacillus (no particular arrangement)
Pink
Gram Negative

Inferences: The Gram Stain is used to differentiate between Gram Positive and Gram Negative bacteria.
The difference between Gram Positive and Gram Negative organisms depends on the structure of the
bacterial cell wall. Gram Positive organisms have high peptidoglycan content, resulting in the ability to
retain stain well. Gram Negative organisms have low peptidoglycan content, resulting in the inability to
retain stain well. Gram Positive organisms will stain purple, while Gram Negative organisms will stain
pink. The Gram Stain also is useful in identifying the morphology and arrangement of microorganisms
when viewing under the microscope (Procedure 4: Leboufe & Pierce, 2013). Unknown Organism #17 had a
bacillus shape, no particular arrangement, and was pink when observed under the microscope. From these
results, it can be concluded that Unknown Organism #17 is a Gram-negative bacillus.
Figure 3: Eosin Methylene Blue agar
Plate (EMB)
After concluding that Unknown
Organism #17 was a Gram-negative
bacterium on the Gram stain, the
organism was plated on an Eosin
Methylene Blue agar (EMB). EMB is
selective for Gram-negative bacteria and
differentiates for lactose fermentation
(Procedure 6: Leboufe & Pierce, 2013).

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(Simms, 2014)

Table 3: Eosin Methylene Blue agar

Table 3 (Refer to Figure 3)
Unknown Organism #17

Observations of Growth on EMB Agar:

Growth; colorless

Inferences: Eosin Methylene Blue agar is a selective and differential medium. It is selective for Gramnegative bacteria and differentiates for lactose fermentation. Because Unknown Organism #17 grew on the
Eosin Methylene Blue agar, the organism is confirmed to be a Gram-negative bacterium. If the bacterium
ferments lactose, coliforms turn metallic green or dark purple. If the bacterium does not ferment lactose, the
organism will remain colorless and take on the color of the medium (Procedure 6: Leboufe & Pierce, 2013).
Unknown Organism #17 was colorless on the Eosin Methylene Blue agar and took on the color of the
medium, which concluded that the organism was a non-lactose fermenter.
PART B: Enzymatic Tests
Table 4: Oxidase Test
Table 4: Oxidase Test Results

Observations
When oxidase reagent was dropped on to filter paper

Unknown Organism #17

containing organism, no reaction occurred.
Inferences: The oxidase test is an enzymatic test that is used to detect the presence of cytochrome c
oxidase. This is detected by the ability of the cytochrome c oxidase to break down cytochrome c and to
catalyze the reduction of cytochrome c by a chromogenic reducing agent, which develop color as they
become oxidized. A dramatic color change indicates the presence of cytochrome c oxidase (Procedure 8:

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Leboufe & Pierce, 2013). When performed on Unknown Organism #17, no color change occurred, which
indicates there is no cytochrome c oxidase present in the organism.

Table 5: Citrate Test:

Table 5 (Refer to Figure 4) Citrate Test
Unknown Organism #17

Observations
Royal blue in color after incubation

Figure 4: Citrate Test

The citrate test was performed to see if Unknown Organism #17 utilized
sodium citrate as a main source of carbon, which would infer that the
organism belonged to the bacterial family Enterobacteriaceae (Procedure
8: Leboufe & Pierce, 2013).
Control Medium

Unknown Organism
#17 medium
(Simms, 2014)
Inferences: The citrate test is performed to identify organisms that utilize sodium citrate as a main source
of carbon and belong to the bacterial family Enterobacteriaceae. When citrate is used as a sole carbon
source, alkaline carbonates and bicarbonates are produced, and the breakdown of ammonium salt produces
ammonia and ammonia hydroxide, which raises the pH. After the test is performed, if the medium is royal
blue, the organism is positive for the utilization of citrate. If the medium remains the same color or is green,
the organism is negative for the utilization of citrate (Procedure 8: Leboufe & Pierce, 2013). Because the
medium turned royal blue when Unknown Organism #17 was tested, the organism utilizes citrate and
belongs to the bacterial family Enterobacteriaceae.
Table 6: Sulfur, Indole, Motility (SIM) Test:

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Table 6: SIM Test for Unknown Organism #17
Sulfur Reduction
Indole Production
Motility

Observations
Negative
Negative
Positive

Inferences: The Sulfur, Indole, and Motility Test (SIM) is used to detect sulfur reduction, indole
production, and motility (Procedure 8: Leboufe & Pierce, 2013). When the SIM test was used on Unknown
Organism #17, the results showed no sulfur reduction, showed no indole production, and showed signs of
motility. From these results, it was inferred that Unknown Organism #17 possessed flagella, did not reduce
sulfur, and did not produce indole.
Urease Activity Test: Identification of Proteus spp.

Figure 5: Urease Activity Test

The Urease Activity test was used to determine if Unknown
Organism #17 belonged to the Proteus genus. Ammonia is
produced when urea is broken down and produces hydroxyl
ions when it reacts with water. This raises the pH. If growth
and color change on the medium occur, the organism produces
urease and is a member of the genus Proteus (Procedure 8:
Leboufe & Pierce, 2013).
(Simms, 2014)
Table 7: Urease Activity
Table 7: Urease Activity: Identification of
Observations
Proteus spp Test on Unknown Organism #17
Urease Activity

No growth or color change; no urease activity

Inferences: The Urease Activity: Identification of Proteus spp Test is used to identify an organisms ability
to produce urease. If an organism is able to produce urease, that organism belongs to the Proteus genus
(Procedure 8: Leboufe & Pierce, 2013). Unknown Organism #17 produced no growth or color change for
this test. From these results, it was inferred that Unknown Organism #17 does not produce urease and was
not a member of the genus Proteus.

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Microbial Genetics Test:

Figure 6: Microbial Genetics Test
The nucleotide sequence for Unknown
Organism #17 was obtained by
performing DNA Extraction via
Freeze/Thaw Cell Lysis and
Polymerase Chain Reaction
(Procedure 11: Leboufe & Pierce,
(AY825036.1, Pan,Y., Chen,W. and Huang)

2013).

Table 8: Microbial Genetics Test

Table 8: Microbial Genetics Test on Unknown
Results
Organism #17
BLAST determined sequence to be Enterobacter
Unknown Organism #17 Nucleotide Sequence

aerogenes. (AY825036.1, Pan,Y., Chen,W. and

Huang,Q)

Inferences: By using the Microbial Genetics Test, the nucleotide sequence for Unknown Organism #17
was obtained. The sequence was inserted into the BLAST search engine, and a matching organism was
determined (Procedure 11: Leboufe & Pierce, 2013). The matching organism that was obtained from the
nucleotide sequence on BLAST was Enterobacter aerogenes. Based on the results from the EMB agar,
Unknown Organism #17 was a non-lactose fermenter. Enterobacter aerogenes is a lactose fermenter. It can
be inferred that either the Microbial Genetics Test or the EMB agar plate produced inaccurate results.

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Table 9: Identification Flow Chart of this Experiment
Identification Tests Performed
Gram Stain

Observations
Gram-negative, bacillus, no particular arrangement
Colonies have a yellow tint, are transparent, have

Growth Characteristics on Nutrient Agar

smooth edges, are small/scattered, are flat and moist

looking
Colorless growth, gram-negative, non-lactose

Growth Characteristics on EMB Agar

fermenter
When oxidase reagent was dropped on to filter
Oxidase Test

paper containing organism, no reaction occurred;

cytochrome c is not present
Royal blue in color after incubation; utilizes citrate;

Citrate Test
belongs to family Enterobacteriaceae
Negative for sulfur reduction, negative for indole
SIM Test
production, positive for motility (possess flagella)
No growth or color change; no urease activity; does
Urease Test
not belong to genus Proteus
BLAST determined sequence to be Enterobacter
Microbial Genetics Test
aerogenes
Discussion:
From all of the identification tests, it can be concluded that Unknown Organism #17 is classified
as Enterobacter aerogenes. Enterobacter aerogenes is a gram-negative bacillus bacteria, is a lactose
fermenter, does not contain cytochrome c, belongs to the family Enterobacteriaceae, possesses flagella,
does not reduce sulfur, and does not produce indole.
Enterobacter aerogenes, along with one other species of Enterobacter, make up for 8.6% of
nosocomial infections (Pathogen Regulation Directorate and Public Health Agency of Canada, 2010).
Enterobacter aerogenes can be found in the gastrointestinal microflora of humans, and reside on urinary
catheters, medical equipment, hygienic materials, dairy products, and soil (Enterobacter aerogenes, 2014,
Rao, 2011, & Enterobacter aerogenes, 2012).
Because this organism is an opportunistic pathogen, and rarely infects healthy individuals, those
prone to obtaining a nosocomial infection from Enterobacter aerogenes are those who have immune

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disorders, those who have been in the ICU, and those who have been affected by invasive instrumentation.
Some infections and problems that can be caused by Enterobacter aerogenes are bacteremia, lower
respiratory tract infection, skin and soft-tissue infections, urinary tract infections, and endocarditits (Rao,
2011).
Enterobacter aerogenes has unique characteristics. Enterobacter aerogenes contains Beta
lactamases, which give this organism great drug resistance. Enterobacter aerogenes also possesses three
mechanisms that provide the bacteria with drug resistance. Enterobacter aerogenes: 1) inactivates enzymes,
2) causes alterations to drug targets, and 3) alters the ability of certain drugs to enter into cells
(Enterobacter aerogenes, 2014). All of these characteristics enable Enterobacter aerogenes to be a
successful opportunistic pathogen and are the reason this bacteria is such a great cause of nosocomial
infections.
Figure 7: Enterobacter aerogenes

(Vetvicka, 2013)
Conclusion:
Throughout the experiment, a major error was made. When performing the Microbial Genetics
Test, the procedure was not followed correctly and all proportions were thrown off. This error could have
caused the nucleotide sequence to be incorrect and could have made the identification of the organism
incorrect; however the proportions were fixed, and the PCR experiment and sequencing produced accurate
results.
The dilemma of this experiment was deciding whether the EMB agar produced inaccurate results
or whether the microbial genetics test produced inaccurate results. Because molecular data is usually more
reliable than biochemical data, it was concluded that the EMB agar produced inaccurate results due to

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contamination, and the organism was identified as Enterobacter aerogenes.
Overall, this experiment was performed carefully and effectively. Five enzymatic identification
tests were performed including an oxidase test, a citrate test, a SIM test, a urease test, and a microbial
genetics test. A Gram stain was performed at the very start of the experiment, and the unknown organism
was plated on EMB agar as well as nutrient agar to determine colony morphology, lactose fermentation
abilities, and to ensure the Gram stain produced accurate results. After performing all of these identification
tests, with the help of Professor Simms, Ms. Sarah, and the identification flowchart provided, it was
concluded that Unknown Organism #17 is Enterobacter aerogenes.

Works Cited
(2014). Enterobacter aerogenes. Retrieved from
http://www.bioquell.com/technology/microbiology/enterobacter-aerogenes/

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(2012). Enterobacter aerogenes. Retrieved from http://www.antimicrobialcopper.com/us/scientificproof/registration-against-bacteria/enterobacter-aerogenes.aspx

Pathogen Regulation Directorate and Public Health Agency of Canada. (2010). Enterobacter aerogenes.
Retrieved from http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/enterobacter-eng.php

Rao, Tumalapalli V. (2011). Enterobacter: An Emerging Nosocomial Pathogen. Retrieved from

http://www.slideshare.net/doctorrao/enterobacter-an-emerging-nosocomial-infection
Procedures:
Leboffe, M.J., & Pierce, B.E. (2013). Brief Microbiology Laboratory Theory and Application. (Second
Edition). Englewood, CO: Morton Publishing.

Figure Citations:
Figure 1:
Rakusin, Alexa. (2014). Enterobacter aerogenes Number Four. Retrieved from
http://modmedmicrobes.wikispaces.com/Enterobacter+aerogenes+number+4
Figure 6:
Pan, Y., Chen, W. and Huang, Q. Retrieved from http://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_56159736.
Figure 7:
Vetvicka, Dr. Vaclav. (2013). Enterobacter aerogenes. Retrieved from
http://www.magma.ca/~scimat/science/E_aerogenes.htm

**Procedures 2, 3, 4, 6, 8, 11 provided by Professor Simms were used throughout the experiment.

**All photos cited (Simms, 2014) were retrieved from Professor Simms.

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