Proteus Vulgaris

(by Erik Lindsay)

After many experiments we can finally conclude that our unknown (#87) is Proteus
vulgaris. First, we tested the purity of our culture by streak plating the bacteria on a
nutrient agar media, as seen.

After seeing that it had positive growth and only one type of bacteria was present, we
performed a gram test. The gram test was repeated many times, until we were satisfied
that our results were correct. After the test we were able to narrow it down to these
remaining species:

Alcaligenes faecalis
Enterobacter aerogenes
Escherichia coli
Proteus vulgaris
Pseudomonas fluorescens
Salmonella pullorum

Under the microscope we could see that our species was pink in color. That meant that it
was gram negative. We used the Bergey’s Manual to figure out which species were gram
positive and which were gram negative. That helped us to eliminate all the gram positive
bacterial species, and focus on these remaining six gram negative species. We were also
able to see that our bacteria appeared to be mainly rod-shaped, but had some coccoid
looking species. Since all of the remaining had rod-like shapes, it just helped to confirm
that our species was one of these six. Here is a look at the gram stain for our bacteria:
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The next test that we performed was a fermentation test. We wanted to see if our species
was an aerobe (nonfermenter) or a facultative anaerobe (fermenter). We inoculated a
sucrose, dextrose, and lactose broth with our bacteria, and incubated them for 24 hours.
Our results were as follows:

Bacterial Species in: Fermented:

Sucrose Positive, +
Dextrose Positive, +
Lactose Negative, -

This experiment was important in narrowing our species down, so we repeated it once
more to confirm our results. Sucrose and dextrose were both fermented by our unknown
bacteria. We saw this because the red broth turned yellow in color. This meant that our
species was a fermenter. This helped us to eliminate the nonfermenters: Alcaligenes
faecalis and Pseudomonas fluorescens. However, our bacteria did not ferment lactose.
Enterobacter aerogenes and Escherichia coli are both lactose fermenters, so we were
able to conclude that they were also not our unknown bacteria. This left us with just two
lactose nonfermenter bacterial species:

Salmonella pullorum
Proteus vulgaris

We used the Bergey’s Manual again to find unique characteristics of the two. Salmonella
pullorum was found to be non-motile, while Proteus vulgaris was motile. We stab
inoculated a test tube of SIM agar with our unknown and incubated it for 24 hours. We
found that the test tube had turned from a reddish color to completely black just below
the surface of the agar. If the black was to have showed up just along the stab line where
we inoculated the agar, it would have meant that our unknown was non-motile. Since the
black color spread throughout the tube, we could see that our bacteria was very motile.
The black color also shows that there was a production of hydrogen sulfide gas, which is
also produced by Proteus vulgaris. These results allowed us to eliminate Salmonella
pullorum, meaning our unknown had to be Proteus vulgaris.

We did not make a final conclusion quite yet though. We wanted to confirm our findings
by seeing if more of the characteristics of Proteus vulgaris matched up with our test
results. Proteus vulgaris is indole positive, liquefies gelatin, reduces nitrate to nitrites,
and does not hydrolyze starch. All of these tests were then performed with our unknown
in order to help confirm that our unknown was actually Proteus vulgaris, and the results
were:
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Experiment performed: Unknown #87 Proteus vulgaris

Indole production Positive, + Positive, +
Reduced nitrates to nitrites Positive, + Positive, +
Liquefies gelatin Positive, + Positive, +
Starch hydrolysis Negative, - Negative, -

In the indole production test we used the same SIM agar that was used for our motility
test. We added 15 drops of Kovac’s Reagent, mixed the solution around, and then let it
sit for five minutes. A red colored band on top of the tube formed, meaning that indole
there was a production of indole. Tryptic nitrate broth was inoculated with our unknown
and incubated for 24 hours. We then added 10 drops of solution A (sulfanilic acid) and
10 drops of solution B (alpha-dimethyl) to the tube and mixed it around. A reddish-pink
color was observed, meaning that our unknown reduced nitrates to nitrites. Next, we
inoculated nutrient gelatin with our unknown and then incubated it for 24 hours. The
gelatin was hydrolyzed by our unknown. Finally, we streaked our unknown onto a petri
dish of starch agar. After 24 hours of incubation we added several drops of Gram’s
iodine to the area of the streak. If the area around the line of growth is clear, starch has
been hydrolyzed, and the test is positive. There was no color change, meaning the starch
was not hydrolyzed and the test was negative.

All of the test results of our experiments matched up with the characteristics of Proteus
vulgaris. We are now confident to confirm that our unknown (#87) is in fact Proteus
vulgaris. Our unknown was very motile, and you can see from this picture that Proteus
vulgaris has many flagella, meaning it is peritrichous.