Assignment #2
Running head: ASSIGNMENT #2
Identification and Characterization of Unknown Gram-Negative Enteric Bacteria
Submitted by:
Kimberly Nelson -S0241236 L15
A paper submitted in partial fulfilment of the
course requirements of BIO 162
Cherie Yoshioka
Lethbridge College
March 25, 2008
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Assignment #2
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Introduction:
The purpose of this experiment is to identify and characterize an unknown gram-negative
enteric bacterium using different bio chemical tests and agents.
Hypothesis: My hypothesis for this experiment is we will find which unknown bacteria
we were given basing it on the bio chemical agents. In the end, our results will match up
correctly, and we will be able to figure out our unknown bacteria ‘F’ without any
contradicting tests.
Materials and Methods:
The materials and methods can be found in the following references:
-“Gram-Negative Intestinal Pathogens” Microbiological Applications, Benson, H.
J., 7ed. Pg 260-263.
-Lab #8 notes.
Results:
In our lab we received an enriched broth culture with an unknown bacterium on it. We
used different tests to figure out which bacteria we had. We observed and documented
the different bacterial growths on the different media from our experiment.
Simmon’s Citrate had no change and stayed a dark green. It tested negative for utilizing
Citrate.
Assignment #2
The SIM test procedure changed from red to a pale yellow. The results showed that it
was positive for motility because there was growth spreading from the stab line. There
was a little layer of pink on the top also where Kovac was added, but the rest is stayed
yellow. There was no black in the medium, so it was negative for sulfur. The reagent
color was unchanged so it was also negative because the tryptophan was not converted
into iodine.
Russel’s Double Sugar (RDS) test started candy pink and turned a bright yellow with
small clusters of bacteria on the slant. Both the butt and slant were yellow, so the
bacterium shows ferments both lactose and glucose.
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Assignment #2
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Urea Test stayed a yellow liquid and was not pink, and had a cloudy layer on the bottom
of the tube. This shows it was negative for urease production.
Enterotube: Glucose was positive and turned yellow. Negative for gas production
because there was no separation. Lysine turned from a yellow to a purple/blue color. This
tells us that this bacterium is able to decarboxylate lysine. Omithine was negative and
was a dull, cloudy yellow color. H2S was negative and a cloudy, peach color with no
black that indicated iodine. Adonitol did not change color and stayed red. It shows that it
cannot ferment arabinose. Lactose was positive. It changed from red to a clear, bright
yellow color. This tells us that this bacterium is able to ferment lactose. Arabinose tested
positive and was a clear, bright yellow colour. Sorbitol was positive too, with an orange,
clear color shown. Voges Prokauer was negative and clear with no color. Phenylalanine
was negative with a green clear color. Urea was negative with a peach, clear color, and
citrate was negative with a green color, and had no clue within it. This showed that citrate
was not utilized.
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Gram stain for our unknown bacteria F showed purple and pink rod shaped bacteria.
However, it is suspected that not all of the crystal violet was washed off because the
bacteria should be all pink/red.
Test
SIM media test
Motility
+/-
Results
+
-Small amount of growth
spreading
form the stab line
-Reagent color is unchanged.
Tryptophan is not converted
to indole
No black in the medium.
Sulfur not reduced.
Indole
-
Sulfur
-
Russel’s Double Sugar
+ both lactose and glucose
Urea Test
Simmon’s Citrate
- for urea production
-
Enerotube
Glucose
Gas production
Lysine
Ornithine
+
+
-
-Started candy pink and
turned a
solid, bright yellow color
throughout the slant with
small clusters on the top of
the slant
-Did not break down urea
-citrate was not utilized and
stayed dark green
-turned yellow
- No separation
-turned a purple/blue color
- was a dull, cloudy yellow
Assignment #2
H2S
-
Adonitol
-
Lactose
Arabinose
Sorbitol
Voges Prokauer
PA
Urea
Citrate
+
+
+
-
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-cloudy peach color, no
black. – for iodine
- color was red, shows it
cannot ferment arabinose.
-clear, bright yellow color
- clear, bright yellow color
- orange, clear color
-clear, no color
- green, clear color
- peach, clear color
-green color, had no blue
within it.
Table 1: Biochemical testing results for unknown bacteria ‘F;’ whether or not the sample
tested positive or negative.
Discussion:
Positive Tests
Glucose
Lactose
Motile
Lysine Decarboxylase
Arabinose
Sorbitol
Explanation of Results
The end result of bacterial fermentation of glucose is an acid;
the shift in pH causes a color change from candy pink to
bright yellow. This tells us this bacterium is able to ferment
glucose.
The end bacterium showed a color change from candy pink to
bright yellow. Fermentation of lactose results in an acidic end
product. This tells us this bacterium is able to ferment lactose.
The result in the SIM test production was pale yellow, and
showed growth spreading from the stab line. This tells us that
this bacterium is motile.
Decarboxylation of lysine results in an alkaline end product
cadaverine, causing a color change from yellow to purple/
blue color. This tells us that this bacterium is able to
decarboxylate lysine.
Fermentation of arabinose results in an acidic end product,
causing a color change from red to yellow. This tells us that
this bacterium is able to ferment arabinose.
Fermentation of sorbitol results in an acidic end product,
causing a color change from red to yellow. This tells us that
this bacterium is able to ferment sorbitol.
Table 2: A description of the positive results, and what caused any color changes.
Assignment #2
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Sources of Error:
1) At any point during the experiment the sample may have been contaminated with
outside material, which would have resulted in false positives. In the end this
would have resulted in us believing we found the right bacteria, when in fact we
found the wrong one.
2) The growth period may have been too long as the lab instructions specified 24
hours, but we left the sample for over a week before checking it. Incubating it to
long may have skewed the results, creating false readings of either negative or
positive results. If bacteria are incubated for too long they may change positive,
however, over time change back. This again would lead us to believe that the
bacteria tested negative when in all actuality it was positive.
3) When stabbing the SIM tube, the wire could have been stabbed too close to the
edge or not all the way to the bottom. This could lead us to believe that the test
was not motile because there was no spreading of bacteria near the bottom, or that
it was motile when the stab mark was already of centre. This could give us a false
reading.
4) When stabbing the double sugar test tube it could have been stabbed too close to
the edge of the tube. This could result in a gas pocket formation between the two
layers of glucose and lactose. This could have prevented the bacteria from
fermenting either the glucose or the lactose and lead to a false negative.
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Conclusion:
We determined from the Enterotube and other test results that our unknown bacterium ‘F’
was escherchia coli.
Lactose (Mac Plate)
Lactose (+)
Pink, muccoid colonies
Indole (+)
Pink surface after adding Kovac’s reagent
Citrate (-)
SIM’s citrate no change, stayed green
escherchia coli
Changes and additions I would do to this procedure to improve the experiment would to
have more than one of the same tests. This would be a confirming match showing
whether or not the test showed negative or positive. This would make the experiment
more exact and less likely for error on figuring out which bacterium you were testing. I
would also have looked at the bacterium after 24 hours, instead of waiting a week. This
would make the tests be more accurate when it comes to determining the bacterium.