The unknown bacteria was identified as Pseudomonas aeruginosa based on several tests. A gram stain showed pink rods that were gram negative and not in chains. A glucose fermentation test with phenol red broth was negative, as the broth remained pink/orange with no gas or color change. An aerobic thioglycolate tube test showed thick growth at the top, indicating it was an obligate aerobe. A blood agar plate initially showed possible alpha-hemolysis but upon further incubation showed definitive beta-hemolysis. Research confirmed that P. aeruginosa has these identifying characteristics of being a gram negative bacillus, producing a grape-like odor, being an obligate aerobe, and causing beta-hemolysis.

1. Rosemary Doerner
BIO 351 LAB
4/27/2015
Unknown Bacteria Report
I obtained the unknown number 79. After conducting a gram stain and looking under the
microscope I saw pink rods which meant it was gram negative bacillus. The rods were not in
chains but rather spread around individually throughout the infected area. After determining the
gram status, arrangement and shape I next conducted a phenol red glucose fermentation test to
see whether or not the bacteria fermented glucose. Next, I conducted a glucose fermentation
test with a phenol red broth tube with glucose. I placed some of the unknown bacteria with my
inoculating loop into the tube and incubated at 37 degrees C for 24 hours. After the amount of
time, I knew that if the tube turned yellow there was glucose being fermented and if there was
presence of a bubble in the Durham tube, there was gas being produced due to the unknown
being able to utilize glucose. However, if it remained pink or orange, then there was no glucose
fermentation.
After the 24 hours, I checked my tube and saw that it was mostly orange/pink and the
top part of the broth was bright pink with a mucous white layer of growth growing on the very
top. I read this test as negative for glucose fermentation due to no yellow color and proceeded
to the next step of the flow chart. I believe the orange and pink colors were due to the alkaline
by product produced and from break down of peptone. I next had to smell the tube in which I
had tested for glucose fermentation. The flow chart stated that if the tube smelt of grapes it
could confirm the unknown to be Pseudomonas aeruginosa. The tube did not smell like grapes
so I proceeded to the next test for confirmation.
The flow chart indicated that if the unknown was Pseudomonas aeruginosa, it would also
be aerobic on a thioglycolate tube test, and would have beta-hemolysis on a blood agar plate.
Knowing this, I next checked for aerobic activity using a differentiation test using a thioglycolate
tube. This test helped differentiate whether the bacteria was obligate anaerobe, obligate aerobe,
facultative anaerobe, microaerophilic or aero tolerant. I sanitized my loop and added my
bacteria to the tube and closed the lid tightly and let incubate for 24 hours at 37 degrees C.
When I checked it the next day it had a thick film of growth on the top which indicated it to be an
obligate aerobe and will only grow on the top where the oxygen is richest. I then conducted a
test to check for hemolytic ability using a blood agar plate. I did a fish tail streak on the blood
agar and left it in the incubator for 24 hours at 37 degree C. After 24 hours I checked the plate

2. and it looked greenish and looked more like alpha-hemolysis than beta-hemolysis. That may
have been due to the fact that my streaks were very thin and close together. There was
however some clearing on the left side. I was unsure about the results so I left the plate in the
incubator for 24 additional hours. When I took it out and looked at it a second time, it was
definitely beta-hemolysis and had a zone of clearing almost like a halo. After conducting all
these tests they all tested positive for Pseudomonas aeruginosa.
Research
Pseudomonas aeruginosa is a gram negative, bacillus bacteria that is part of the Pseudomonas
genus and Pseudomonadaceae family (Lessnau, 2014). The bacteria has a specific
arrangement of the rods that are spread out and not in chains or clusters and has a distinct odor
similar to grapes (Todar, n.d.). This bacteria is found in water, soils, leaves, animals (Todar, n.d.)
as well as on contaminated hands, equipment surfaces, and unclean pools (“Pseudomonas
aeruginosa”, 2014). Pseudomonas aeruginosa is an aerobic bacteria, which means it only
grows in the presence of oxygen, however, in specific cases it can grow anaerobically such as
in the presence of NO3 (Todar, n.d.). This bacteria is highly motile and can withstand growth at
temperatures up to 42 degrees C (“What is Pseudomonas”, n.d.). The motility make it easy to
spread and swim with its flagella around wet or moist environments and its ability to grow at
high temperatures make it hard to control. Pseudomonas aeruginosa also have very limited
nutritional needs which is why they can grow almost anywhere (Lessnau, 2014). Pseudomonas
aeruginosa also does not ferment and has beta hemolytic ability. This bacteria is normally non-
pathogenic on individuals who are healthy and will grow on normal flora of about 0-24% of
people who are not hospitalized (CDC, 2014). P. aeruginosa, however, likes to attack victims
who are immunocompromised. P. aeruginosa is the cause of numerous Pseudomonas
infections widely found in hospital settings today, where it targets its highest amount of victims
(Todar, n.d.). According to the Center of Disease Control, there are around 51,000
Pseudomonas infections a year (“Pseudomonas aeruginosa”, 2014). When this happens,
victims can suffer infections in any part of the body including bone, ear, urinary tract, skin, heart,
respiratory tract and GI tract (Lessnau, 2014). When patients in hospitals contract these
infections it can easily spread and become deadly due to their weakened immune system.
Healthy individuals will not usually develop an infection from Pseudomonas aeruginosa, but if
they do, their symptoms are much milder and consist of ear infections, skin rashes or eye
infections (Pseudomonas, aeruginosa”, 2014). The treatments of these infections are very
complicated. The obvious first choice would be antibiotics, however, Pseudomonas aeruginosa

3. are resistant to many antibiotics (Allaby, 2003.). This is likely due to its ability to live anywhere
and have low nutrient needs. Since there is no vaccine for this bacteria, it is important to take
preventative steps in order to not get an infection. Washing hands, not swimming in dirty water
sources, and disinfecting surfaces are some ways to limit the chances of getting infection from
this bacteria.

4. Citations
Allaby, M. (2003). Pseudomonas. Retrieved April 29, 2015, from
http://www.encyclopedia.com/topic/Pseudomonas.aspx
Lessnau, K. (2014). Pseudomonas aeruginosa Infections. Retrieved April 29, 2015, from
http://emedicine.medscape.com/article/226748-overview
Pseudomonas aeruginosa in Healthcare Settings. (2014, May 7). Retrieved April 29, 2015, from
http://www.cdc.gov/hai/organisms/pseudomonas.html
Siegrist, J. (2015). Pseudomonas Media and Tests. Retrieved April 29, 2015, from
http://www.sigmaaldrich.com/technical-documents/articles/analytix/pseudomonas-media.html
Todar, K. (n.d.). Pseudomonas. Retrieved April 27, 2015, from
http://textbookofbacteriology.net/pseudomonas.html
What is Pseudomonas aeruginosa? (n.d.). Retrieved April 29, 2015, from
http://www.ehagroup.com/resources/pathogens/pseudomonas-aeruginosa/