Identification of Enterobacter aerogenes
by Different Microbiological Methods
Microbiology 225-013
Jennifer Salmorin| 23 April 2012
Title:
Identification of Enterobacter aerogenes by Different Microbiological Methods
Abstract:
Given an “unknown” culture in a TSA agar slant, the class objective was to identify the unknown
organism by using various laboratory procedures and experiments that would assist in arriving
at an educated conclusion regarding the Genus species. The first procedure involved preparing
a Gram’s stain to identify if the organism was gram positive or negative and determine the cell
morphology. After examining the stained organism under the microscope under the oil
immersion lens (100X), the organism’s cell shape could be identified as gram positive or
negative. The organism was identified as a gram negative, based on its red stain, and bacilli,
based on the rod shape. A streak plate was also prepared by inoculation and incubation to
display the colony pattern of the organism. After identification of the gram negative bacilli, the
lactose fermentation test was performed to determine the bacteria’s ability to ferment lactose.
After inoculation and incubation of the lactose broth, the organism was determined to be
lactose positive due to its yellow coloring after the addition of a phenol red indicator. Once this
test was complete, and the result identified, the indole and H2S test was performed
simultaneously to determine the organism’s ability to produce indole and reduce sulfur into
H2S, respectively. A SIM deep was inoculated with the organism and incubated for 48 hours.
After incubation, a Kovac’s reagent was added to the SIM deep, and a negative result was found
for both tests (yellow for indole test and no black precipitate present for H2S test). After this
result was determined, and the organism narrowed down to two, a final methyl red test was
performed to test for the acid end products from glucose fermentation and to ultimately
identify the organism. A glucose broth was inoculated with the organism and 48 hours after
incubation, a methyl red indicator was added to determine the identity of the Genus species.
The test result was negative due to the yellow coloring of the broth after the addition of a
methyl red indicator. The Genus species was therefore identified as an Enterobacter
aerogenes.
Introduction
The Enterobacter aerogenes are most commonly identified as hospital acquired infections, also
known as nosocomial infections. This gram negative bacteria is a rod shaped (bacilli) and exists
in soil, water, dairy products, as well as resides in the natural flora in the gastrointestinal tract
of animals and humans alike (2).
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Below is a view of the bacteria under a microscope using a Gram’s stain (2):
The majority of these infections are attributed to accidental transfer of bacteria to the patient
during surgery or transfer of infection through urethral catheters. They are opportunistic
pathogens that infect patients with compromised immune systems (2). They are responsible
for various infections that include bacteremia, lower respiratory tract infections, skin and softtissue infections, urinary tract infections, endocarditis, intra-abdominal infections, and more
(1). Fifty percent of septicemia and seventy percent of urinary and intestinal infections are
caused by these bacteria.
A major characteristic of these bacteria is that they are extremely resistant to β-Lactam
antibiotics, fluroquinolones, chloramphenicol, tetracycline, and kanamycin; this is due to the
mutations that encode porins and membrane efflux pumps that pump out the antibiotics before they
have a chance to destroy the bacteria (2 and 3). The resistance of these bacteria to antibiotics and
other drugs has lead to outbreaks of infections in intensive care units in Belgium, France, Austria,
and the United States (2).
There are several ways to avoid the infection of Enterobacter aerogenes. Many of these methods are
left up to healthcare providers caring for patients in a hospital setting. One way is to assure that all
instruments used during surgical procedures are sterile. Another way to reduce the spread of these
bacteria is removing catheters and venous needles and tubing and placing them in new locations (2).
Avoiding third generation cephlasporins is also crucial because resistant strains can produce (1).
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Materials and Methods
Gram Staining Materials (5):






Inoculating loop, glass slides, Bunsen burner, Distilled water.
Crystal Violet (Hucker’s) — Solution A of crystal violet (90% dye content) was made by
dissolving 2.0 g of crystal violet in 20 ml of 95% ethyl alcohol. The solution B was made
of 0.8g Ammonium oxalate in 80 ml of distilled water and finally the solution A and B
was mixed together.
Gram’s Iodine — Iodine 1.0g, Potassium iodide 2.0g was dissolved in 300 ml of distilled
water.
95% Ethyl alcohol — Ethyl alcohol (100%) 95.0 ml and 5 ml of distilled water.
Safranin — Safranin O 0.25 ml, Ethyl alcohol(95%) 10 ml and 100 ml of distilled water.
The Gram’s reagents were aliquoted in small dispensing squeeze bottles for laboratory
use.
Lactose Fermentation Test Materials (4):
Lactose broth with phenol red (pH 7.3):




Trypticase:
Sucrose:
Sodium Chloride:
Phenol red:
10.0 g
5.0 g
5.0g
0.018 g
The media was aliquoted in 5ml per test tube and autoclaved at 15 lbs pressure for the
minimum of 15 minutes.
Indole Production Test / Hydrogen Sulfide Test Materials:
SIM Agar Deep (4):





Peptone
Beef extract
Ferrous ammonium sulfate
Sodium thiosulfate
Agar
30.0 g
3.0 g
0.2 g
.025 g
3.0 g
4
Kovac's reagent: (per liter) (6)



p-Dimethylaminobenzaldehyde
Amyl Alcohol
Hydrochloric acid
50.0g
750.0 ml
250.0 ml
Other Materials (8):

Bunsen burner, inoculating needle, test tube rack and glassware marking pencil
Methyl Red Test Materials:
MR-VP Broth (3):



Peptone
Dextrose
Potassium phosphate
7.0 g
5.0
5.0 g
Methyl Red Indicator (9):



Methyl Red
.10 g
Ethanol, 95%
300 mL
Distilled water to make 500mL
Other Materials (8):

Bunsen burner, inoculating loop, test tubes, and glassware marking pencil
Experiments
An agar slant containing an unknown organism with an associated number, 9, was received in
the lab. Flow charts were distributed to track the experiments performed for the organism.
Gram’s Staining:
A differential staining technique called Gram’s staining was performed to identify whether the
bacterial cells were gram positive or negative, as well as to determine the shape of the cells.
First, a smear of the organism was made on a clean glass slide by aseptic technique. This
technique involved placing a drop of distilled water onto a slide and then transferring the
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organism onto the glass with a sterile inoculating loop. The smear was air-dried and then heat
fixed by quickly passing through the Bunsen burner flame. The smear was flooded with crystal
violet for one minute. After a minute was up, the crystal violet was washed off with tap water.
Next, the smear was thoroughly rinsed with Gram’s iodine mordant for one minute and then
the Gram’s iodine was washed off with tap water. The slide was then gently decolorized with
95% ethyl alcohol by slowly dripping the alcohol onto the slide until it ran mostly clear. Once
again, the slide was washed with tap water. The slide was then counterstained with safranin
for 45 seconds and then washed with tap water. The slide was blotted dry with bibulous paper
and examined under oil immersion under the microscope (10).
Microscope Observation:
After completion of the Gram’s stain procedure, the slide was observed under an Olympus
CX21/31 binocular microscope under the oil immersion objective (100X) to determine whether
the organism was gram positive or gram negative, as well as to identify the cell’s shape and
morphology.
Lactose Test:
A lactose broth was inoculated with the organism using aseptic techniques and incubated at 37
degrees Celsius for 36 to 48 hours. After 48 hours, a phenol red indicator was added to the
broth and the culture was examined for lactose fermentation (11).
MacConkey (MAC) Agar Plate and Trypticase Soy Agar (TSA) Plate Streak:
The unknown organism was streaked with an inoculating loop using aseptic techniques to show
colonization in both a MAC plate as well as a TSA plate. After the first streak in the upper
quadrant of both the TSA and MAC plates was made, the plate was rotated 90 degrees and
streaked again once the loop was re-flamed. It was then rotated another 90 degrees and
streaked again after re-flaming the loop. It was rotated another 90 degrees and streaked for a
final time (12).
Indole/ Hydrogen Sulfide Test:
The indole and hydrogen sulfide tests were able to be performed simultaneously. A SIM deep
agar was stabbed with an inoculating needle containing the organism using aseptic technique.
The tube was incubated for 48 hours at 37 degrees Celsius. After 48 hours of incubation, the
tube was removed and ten drops of Kovac’s reagent was added to the culture. It was examined
for color change at the top of the culture for indole production (13). It was also examined for
black coloration along the stab line to determine if it was motile and if it had any H2S
production (14).
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Methyl Red Test:
An MR-VP broth was inoculated with the organism using aseptic technique. The tube was
incubated for 48 hours at 37 degrees Celsius. Five drops of methyl red indicator was added to
the culture and the color was examined for glucose fermentation (13).
Results and Discussion/Conclusion
The Gram’s stained slide displayed pinkish-red stubby rods when observed under the oil
immersion lens (100X) of the microscope. This microscopic observation indicated that the
organism was a gram negative bacilli (4). Following the Gram Negative Bacilli flowchart, a
lactose test was performed next.
After performing the lactose test, a phenol red indicator was added, and it was determined that
the organism was negative for lactose fermentation due to the red coloring (or no change in
color) (11).
Next, the indole and hydrogen sulfide production tests were performed simultaneously in the
same tube. After 48 hours of incubation, ten drops of Kovac’s reagent was added to the
culture. It was examined for color change at the top of the culture (where the Kovac’s reagent
was added) for indole production (13). After the addition of the Kovac’s reagent, the coloring
remained yellow (no color change occurred). This indicated that the substrate tryptophan was
not hydrolyzed and tested negative for indole production. The same tube was also examined
for black coloration along the stab line to determine if it was motile and if it had any H2S
production. The absence of black coloration indicated that the organism was non-motile and
that no sulfur was reduced into hydrogen sulfide (H2S), resulting in a negative reaction (14).
Finally, a methyl red test was performed to identify the presence or absence of glucose
fermentation. After inoculation and incubation, a methyl red indicator was added and the
culture turned from red to yellow, indicating a negative result for glucose fermentation (13).
Once this test was completed and the results examined, it was determined that the unknown
organism was Enterobacter aerogenes, according to the Gram Negative Bacilli flowchart.
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