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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 anutrient agar media, as seen.

After seeing that it had positive growth and only one type of bacteria was present, weperformed 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 Bergeys Manual to figure out which species were grampositive 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 coccoidlooking 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 asucrose, 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 unknownbacteria. 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 andEscherichia coli are both lactose fermenters, so we wereable 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 Bergeys Manual again to find unique characteristics of the two. Salmonellapullorum 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. Wefound 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 theblack 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 ofProteus 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 resultswere:

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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 Kovacs 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 unknownand 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, weinoculated 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 Grams

iodine to the area of the streak. If the area around the line of growth is clear, starch hasbeen 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 ofProteusvulgaris. 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.