Elizabeth Berry
Wednesday at 3:00 p.m.
Unknown #640
Salmonella enterica
Gram Stain
This is a picture of a gram stain using a brightfield microscope on a 100x objective lens
producing an image that has a total magnification of 1000x. This differential stain shows
clusters of gram-negative bacilli organisms (The organism has a clearer image and appears
more pink in person).
Streak Plate-TSA
The organism grew the best on a TSA plate incubated at 37 degrees Celsius. These colonies
appear to be a yellowish, white color. They also appeared to be round and flat with smooth
margins. They seem to be in the middle of the spectrum as far as diameter—not extremely
large nor extremely small but more in the middle.
Biochemical Tests
Test
Reagent/Media Enzyme/end
product
3% KOH
Gramnegative cells
will lyse in
the KOH
solution and
release their
DNA
Interpretation
Catalase
Hydrogen
peroxide
Catalase
enzyme will
break down
hydrogen
peroxide to
oxygen and
water if it is
present
Bubbles
formed
indicating the
catalase
enzyme is
present in this
organism
Oxidase
Oxidase reagent
Test for the
presence of
the enzyme
cytochrome
oxidase
There was no
color change
indicating the
organism is
negative for
the enzyme
KOH
DNA strings
were observed
confirming the
organism is
gram-negative
Picture
Methyl Red
MRVP broth
tube/methyl red
indicator
reagent
The ability to
produce and
maintain
stable acid
end products
from glucose
fermentation
Turned to a
pinkish red
color
indicating the
organism
produced acids
as an end
product from
fermentation
Voges Proskauer
MRVP broth
tube/ VP-A and
VP-B reagent
(40% KOH)
Produce
neutral end
product 2,3
butanediol
Color did not
change to red
indicating
organism could
not produce
neutral end
product
Phenol Red
Broth media
Fermentation Fermentation
Fermentation: with arabinose
of arabinose of arabinose
Arabinose
and pH
occurred
indicator phenol
because color
red
changed to
yellow
Phenol Red
Broth media
Fermentation: with lactose and
Lactose
pH indicator
phenol red
Fermentation Remained
of lactose
close to
original color
(red) which
indicates
lactose was not
fermented
Phenol Red
Broth media
Fermentation Fermentation
Fermentation: with mannitol
of mannitol
of mannitol
Mannitol
and pH
occurred
indicator phenol
because color
red
changed to
yellow
Phenol Red
Broth media
Fermentation Fermentation
Fermentation: with sucrose
of sucrose
did not occur
Sucrose
and pH
because the
indicator phenol
color stayed
red
closer to the
original uninoculated red
color.
Citrate
Alkaline slate
tube
Ability to
utilize citrate
and ammonia
salts will
grow on this
media
Color changed
from green to
blue indicating
the organism
utilized citrate
as a sole
carbon source
and ammonia
as a sole
nitrogen
source
Urea
hydrolysis
Slant tube with
pH indicator
phenol red
Enzyme
urease’s
ability to
hydrolyze
urea yielding
ammonia
Remained
close to the
original color
indicating the
enzyme urease
was not
present
SIM-motility
SIM tube
and Hydrogen
Sulfide
production
Hydrogen
Sulfide
production
and growth
outward
from the stab
line
SIM-Indole
Indole
production
SIM
tube/Kovac’s
reagent
Blackening
covered the
tube indicating
Hydrogen
Sulfide was
produced. It
also indicates
that this
organism is
motile because
it radiated
away from stab
line.
Color remained
black (instead
of change to
red) indicating
this organism
did not
produce indole.
After Kovac’s was added
Discussion Page
On April 9, 2014, I was assigned unknown number 640 and had to determine the
type of unknown bacteria growing in the slant agar tube. I performed my first set of tests,
which was a gram stain, a KOH test, an oxidase test, and a catalase test. These tests lead to
the gram-negative family Enterobacteriaceae. To finish up my first day, I incubated two
new TSA plates that were streaked with the unknown bacteria both at 37 degrees Celsius.
I returned to the lab on April 11, 2014, allowing my organisms to have incubated for
48 hours. Both TSA plates had growth present. Unfortunately it was a Friday, so the only
test I could start was the MRVP test due to a 72-hour incubation period. I also had to streak
a new TSA plate. The next week I finished the MRVP test and had to decide which tests
would be suitable to perform because the results of the MRVP test. I chose a SIM test,
Phenol Red tests (Arabinose, Lactose, Sucrose, and Mannitol), a Citrate test, and a Urea test.
The SIM test and the Phenol Red Mannitol test verified that the unknown organism was
Samonella enterica. The other tests (remaining Phenol Reds, Citrate, and Urea) were
performed to confirm that this organism was in fact Samonella enterica.
The only test results that didn’t come out right were the Citrate test and the Phenol
Red Lactose and Sucrose. The Citrate test’s color changed making it positive when it should
have been a negative result. This could be because it was incubated for 48 hours instead of
24 hours due to the lab being closed. As far as the Phenol Red tests, both sucrose and
lactose were a pinkish, orange color. This made me hesitant to say it was negative, but
because it was closer to the original color than a yellow color I determined that it was a
negative result.
Throughout this process of identifying my unknown, I have expanded my
knowledge of how easy and fun it can be to identify a single organism from a number of
possible tests and observations. This overall process showed me why it is important in our
everyday lives as well. An example of this would be when the doctor takes a swab of your
throat and sends it off to a lab to determine what microbe is present. Since I do want to go
into the health care field, I found that my unknown, Salmonella enterica, is associated with
food poisoning in several countries around the world. This is important to know because if
food is prepared improperly than you could be dealing with the repercussions of this nasty
little organism.
Works Cited
http://www.sva.se/en/About-SVA/Salmonella-website/General-facts-about-salmonella/
Gram
Stain
Negative
Enterobacteriaceae
Pseudomonadaceae
Rods
Morphology
Catalase
Positive
Pseudomonadaceae
Enterobacteriaceae
Oxidase
VogesProskauer
Negative
E. coli
Negative
Streptococcaceae
Cocci
Streptococcaceae
Staphylococcaceae
Catalase
Positive
Staphylococcaceae
Positive
Pseudomonadaceae
Negative
Enterobacteriaceae
Negative
Samonella
enterica
Klebseilla
pneumoniae
Escherichia
coli
Proteus
mirabilis
Proteus
vulgaris
Morphology
Rods
Bacillaceae
Cocci
Enterobacteriaceae
Pseudomonadaceae
Negative
Positive
Streptococcaceae
Staphylococcaceae
Bacillaceae
Positive
Klebsiella
pneumoniae
Enterbaeter
aerogenes
Serratia
marcescens
SIM
H2S
production
Negative
Klebseilla
pneumoniae
Enterobaeter
aerogenes
Serratia marcescens
Methyl
Red
Positive
Samonella
enterica
Escherichia
coli
Proteus
mirabilis
Proteus
vulgaris
Positive:
S. enterica, P. mirabilis, P. vulgaris
Negative
Negative
S. enterica, P.
mirabilis
Negative
P. mirabilis
SIM
Motility
SIM
Indole
Phenol Red
Fermentation:
Mannitol
Positive
S. enterica, P. mirabilis, P.
vulgaris
Positive
P. vulgaris
Positive
S. enterica