Unknown Lab Report
Emily Freeman
Tuesday, 1-2:50 PM
Organism number: 407
Organism: Salmonella choleraesuis
Organism identity: Salmonella choleraesuis
Gram Stain: The cells were pink (gram negative) bacilli. The cells were not joined
together, but all of them were single rods. A bright-field microscope was used on
1000X magnification.
Streak Plate: The culture was streaked onto a TSA plate and incubated each time
for approximately 24 hours in 37 degrees Celsius. There were colorless, isolated
colonies of S. choleraesuis in quadrants two and three. There were few colonies in
quadrant four on the plate used for the photo. It was never an issue obtaining
isolated colonies on the TSA plate.
Biochemical Tests:
Test
Reagent/Media
Enzyme/End
Product
Determines the
presence of the
catalase enzyme.
Catalase
Hydrogen peroxide
Oxidase
3 drops of oxidase
reagent
KOH
3% KOH solution
H2S Production
SIM tube medium
Motility
SIM tube medium
Determines
motility
Indole
SIM tube medium;
3-4 drops of
Kovac’s reagent
To determine
indole
production.
Determines
whether the
enzyme oxidase
cytochrome is
present.
Gram negative:
stringy
substance
Gram positive:
no reaction
Determines
whether H2S is
produced or not
End
Product/Interpretation
Small bubbles were seen
when the hydrogen
peroxide was added.
This means the catalase
enzyme broke down
hydrogen peroxide into
water and oxygen.
The cotton swab did not
turn purple, meaning it
does not have the
enzyme.
The bacteria became
stringy, meaning it is
gram negative.
Blackening along the
stab line occurred
showing there was H2S
production.
The blackening of H2S
production moved
beyond the stab line to
the edges of the tube
indicating motility.
There was no color
change to red, meaning
no indole was produced
TSI
(slant/butt)
Triple sugar iron
agar
MacConkey
MacConkey Agar
EMB
EMB agar
Mannitol
Mannitol- Phenol
Red medium
Arabinose
Arabinose- Phenol
Red medium
Sucrose
Sucrose- Phenol
Red medium
Determines
whether bacteria
can reduce sulfur
and ferment
carbohydrates.
Shows acid
and/or alkaline
production as
well as gas and
H2S production.
This determines
whether the
organism grow
with bile salts
and crystal
violet.
Show that
organisms are
able to grow
with eosin Y and
methylene blue.
This test
determines if the
organism can
ferment a
carbohydrate.
This test
determines if the
organism can
ferment a
carbohydrate.
Determines
whether the
organism can
ferment the
carbohydrate.
Acid production (yellow
color change) occurred
in the butt of the agar
and alkaline (pink color
change) occurred in the
slant.
The colonies were
colorless meaning they
did not ferment lactose
or sucrose and produce
acid.
Colorless colonies were
seen meaning there was
no sucrose or lactose
fermentation and no acid
produced.
The medium turned from
red to yellow because
acids are produced when
the organism ferments
the carbohydrate.
The medium turned from
orange to orange/yellow
because acids were
produced due to the
fermentation of
arabinose.
The medium remained
red because it was not
able to ferment sucrose.
Gram
Stain
Positive:
Streptococcaceae
Staphylococcaceae
Bacillaceae
Negative:
Enterobacteriaceae
Pseudomonadaceae
Morphology
Morphology
Rods:
Enterobacteriaceae
Pseudomonadaceae
Cocci
Cocci:
Streptococcaceae
Staphylococcacea
e
Rods:
Bacillaceae
Catalase
Catalase
Positive:
Pseudomonadaceae
Enterobacteriaceae
Negative
Positive:
Staphylococcaceae
Oxidase
Negative:
Enterobacteriaceae
Positive:
Pseudomonadaceae
H2S Production
Positive:
Salmonella choleraesuis
Proteus mirabilis
Proteus vulgaris
Negative:
Klebsiella pneumoniae
Enterobacter aerogenes
Enterobacter cloacae
Escherichia coli
Citrate
Positive:
Proteus mirabilis
Negative:
Salmonella choleraesuis
Proteus vulgaris
Mannitol
Negative:
Streptococcaceae
Positive:
Salmonella choleraesuis
Negative:
Proteus vulgarus
Indole
Indole
Negative:
Salmonella choleraesius
Positive:
Proteus vulgaris
Discussion:
Overall, the identification had few difficulties. Initially, when doing the mannitol
and sucrose tests, they became mixed up because they were not labeled right away.
This gave me unclear results. Because of confusing results, I redid the tests. The
results from the second testing were accurate. Another issue was repeated false
negative catalase tests. This test had to be repeated twice in order to see bubbles.
The second time the test was done, the bubbles were very small and hard to see.
After narrowing my unknown down to the Enterobacteriaceae family, I streaked an
EMB and MacConkey plate to confirm my unknown was gram negative (due to the
unclear catalase test). Using the SIM tube and determining H2S production allowed
me to narrow down to three organisms. The citrate test was negative, which
eliminated Proteus mirabilis. The phenol red tests (sucrose, arabinose, and
mannitol) differentiated Salmonella choleraesuis and Proteus vulgaris. I completed
the Indole test to confirm that my organism was Salmonella choleraesuis.
I learned it is very important to label all of my tests immediately to save myself from
confusion. I also learned it is important to use fresh culture when doing the
biochemical tests because false negatives can occur (as I saw with the catalase test).
S. choleraesuis can be found in the egg yolk of poultry eggs. It can also be in swine
and cattle meat. Proper food storage and cooking can kill the bacteria to make it
safe for eating. As a Nutrition and Dietetics major, this information is helpful
because I will be educating people on proper eating and this is crucial to preventing
infections.