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Proteus vulgaris | Unknown Lab Report, Microbiology

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Tony Rann


Fall 2013



Microbiology in simplest terms is the study of microorganisms that may or may not be present to the naked eye. These microorganisms can have remarkable abilities such that we cannot fathom. In order to begin to understand them, we must learn the basics. Some of these organisms have distinct qualities that set them apart from one another, such as the exposure to certain environments. Through out the semester in the laboratory, we are able to encounter some of the few microorganisms that we as humans have come into contact with. With the knowledge gained from the sessions in the laboratory, we can now integrate what we have learned to the process of finding out the unknowns given.

Materials and Methods

The unknown specimen was given out by the Professor. The test tubes were labeled number 122 and Alt 5. The primary objective was to isolate two different colonies within the test tube. First off, a streak plate must be done on a nutrient agar plate. After 48 hours of incubation, nutrient agar plate showed two distinct colonies.  Once the two colonies were found, they were isolated on separate agars for growth.

After another 48 hours, there were two different unknown bacterium growing on the two different nutrient agar plates used. Lastly, to determine which is a gram negative and gram positive, a gram stain test was used.

Unknown number 122 came out to be gram positive. Under the microscope unknown number 122 showed to be blue-colored rods. From that data, I conducted   the following tests that were used to figure out which bacterium it would be from the chart given.

Gram Positive Test

-Starch Test

-Oxidase Test

-Casein Test

-Maltose Test

Alt 5 under the microscope showed a red color indicating that it is a gram negative bacterium. The shaped of this bacterium was rounded and ball shaped. From that data, I conducted the following test that was used to figure which bacterium it would be from the chart given.

Gram Negative Test

-Simmons Citrate Test

-Methyl Red Test

-Nitrate Test

-Mannitol Test


GRAM POSITVE #122(Gram Positive)

ALT 5 (Gram Negative)

Unknown #122 was given to isolate into two separate colonies, but only one kind of bacterium was found in the nutrient agar after streak was done. Gram stain showed this bacterium to be a gram-positive bacteria, it showed the blue colored rods.

Alt 5 was given as a substitute for a gram-negative bacterium, due to no growth in original test tube (#122). The gram stain showed red colored rods.

The oxidase test was performed on unknown #122 gram positive, showed to be positive. Starch test was performed; it showed a clearing on the agar, indicating a positive result. Lastly casein test was performed, test showed positive result for the break down of casein.

The Simmons citrate test was performed, no color change meaning negative result. Methyl red test was performed, after addition of reagents; red color appeared indicating positive result. Nitrate test was performed, test showed to be positive after the reagents were added. Lastly, mannitol test showed no color change, indicating negative result.

Table 1. Gram Positive Test


Test Purpose Reagents/agar Observations Results
Gram Stain To determine the Gram Reaction of the given bacterium Gram crystal violet, Gram iodine, Gram decolorizer, Gram safranin Blue colored rods under the miscroscope Blue rods= Gram postive
Oxidase Test To determine the presence of cytochrome oxidase Oxidase paper Color change from purple/black Positive oxidase test
Starch Test To determine the production of amylase Starch agar Clearing around streak of bacterium postive
Casein Test To determine if bacterium produce casease which breaks down the milk protein casein Milk agar Bacterium produces casease which hydrolyzes casein Positive result for break down of casein


Table 2.  Gram Negative Tests


Test Purpose Agars/Reagents Observations Results

Gram Stain Test

To determine the Gram Reaction of the given bacterium Gram crystal violet, Gram iodine, Gram decolorizer, Gram safranin Red colored rods found under microscope Red colored rods= gram negative
Simmons Citrate Test To determine if organism can use citrate as its sole carbon source Simmons Citrate tube Green color in tube suggest reaction with bacterium is negative Negative
Methyl Red Test To determine if a bacterium that can produce a mixture of acids as a result in glucose fementation Methyl red After adding the methyl red reagent, the liquid slowly turn a red color Postive
Nitrate Test To determine if an organism can reduce nitrate to nitrite or other nitrogenous comounds Nitrate 1 and 2


Color change after the addition of the reagents of nirate 1 and 2, but none after the addition of zinc Postive
Mannitol Test To determine if bacterium can ferment mannitol Mannitol test tube No color change indicatate bactiruim does not fement mannitol Negative


Discussion / Conclusion 

For the Gram positive bacteria, I ended up with the my unknown #122 being Bacillius cereus. I had my times of mistakes trying to find out these bacteria from the rest but I stuck to the fundamental basics of the lab manual and believed in myself. The following test below shows my journey towards finding my unknown.

The starch test was used to identify which bacterium can produce the enzyme amylase. The result, showed a clearing or halo ring around the streak of bacterium. That indicated a positive result, thus eliminating: Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis. This left only two possible bacterium: Bacillus cereus and Bacillus subtilis.

The oxidase test was used to show if the organism produced cytochrome oxidase, with the addition of the oxidase reagent. The result, which presented the color blue after 15 minutes. After the data was collected, this test showed that this could only be Bacillius cereus, as it came out to be a positive reaction. This data helped eliminate the remaining bacteria, Bacillus subtilis.

The casein test was used to show which organism would produce the enzyme casease, which hydrolyzes the milk protein casein. In this test, the result showed a clearing around the streak of bacteria indicating that it was a positive result. Thus confiming the unknown specimen to be Bacillius cereus. 

For the gram-negative bacteria, I had trouble with this from the beginning. The gram negative in my original tube (unknown #122) would not grow or even show up in the nutrient agar. So I was given a tube that was a gram negative bacteria by Professor Snaric, from that moment on, every went by swiftly as I tried to find out my unknown (ALT 5).

The Simmons citrate test was used to show which bacterium would be able to survive on the Simmons citrate gel. This test allows bacterium that can use citrate as its sole carbon source. For a positive reaction, the color of the gel within the citrate tube must change color from green to blue. As for a negative result, no color change. The bacterium was added to the citrate and left to incubate for 48 hours, results showed a negative result. This eliminated three other bacterium: Klebsiella pneumonia, Enterobacter aerogenes, and Pseudomonas aeruginosa, leaving E. coli and P. vulgaris.

The methyl Red test was used to show which bacterium would be able to produce a mixture of acids that would ferment glucose. A positive result is a red color in the liquid, and a negative result would be a yellowish color. After the test was done, the result positive after the addition of the reagent. This eliminated two bacterium: Enterobacter aerogenes and Pseudomonas aeruginosa, leaving E. coli, P. vulgaris and K. pneumonia. 

The nitrate test was used to show if a bacterium would be able to reduce nitrate to nitrite or other nitrogenous compounds. After the addition the nitrate reagents the liquid turned from yellow to red, indicating a positive result. This test eliminated E. coli, leaving the others showing a positive reaction.

The mannitol test was used to show if a bacterium could ferment mannitol. For this test, I was able to finalize the result of my unknown to be Proteus vulgaris.

Proteus species are part of the Enterobacteriaceae family of gram-negative bacilli. Proteus organisms are implicated as serious causes of infections in humans, along with Escherichia, Klebsiella, Enterobacter, and Serratia species. (Struble 2013) Proteus species are most commonly found in the human intestinal tract as part of normal human intestinal flora, along with Escherichia coli and Klebsiella species, of which E coli is the predominant resident. Proteus is also found in multiple environmental habitats, including long-term care facilities and hospitals. In hospital settings, it is not unusual for gram-negative bacilli to colonize both the skin and oral mucosa of both patients and hospital personnel. Infection primarily occurs from these reservoirs. However, Proteus species are not the most common cause of nosocomial infections. (Struble 2013)



  1.  McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General Microbiology.
  2. Struble,Kelly. 25 March 2013. Proteus Infections. Medscape. University of Oklahoma College of Medicine. Retrieved December 2, 2013


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