Unknown Lab Report (Pseudomonas aeruginosa)
by Kiley Vujnich
Microbiology
Fall 2012
INTRODUCTION
The purpose of this lab was to identify two unknown bacteria, a gram positive and a gram negative, from a mixed culture. It is important in the medical field to be able to identify unknown bacteria to help treat a patient by knowing the source of the illness. This identification was done using methods learned during this semester in Microbiology.
MATERIALS AND METHODS
A mixed culture unknown labeled #122, was given out by the instructor. The methods used for identification have been learned during the semester and applied to this unknown. Procedure for each of the tests used were found in the lab manual, and followed according to those directions. The first procedure that needed to be completed was an isolation plate for each of the unknown bacterium. After determining the shape and differences in the cell wall via the Gram stain, specific tests were done to each of the unknowns. These tests were chosen based on the information given by the lab manual and observations from labs performed throughout the semester.
After incubation, both of the NA plates turned green indicating growth of one bacterium, not two. From colonies produced on one of the NA plates, an Eosin-Methylene Blue Agar plate (EMB) was used to isolate the Gram negative bacteria since EMB plates inhibit the growth of Gram positive bacteria. Two more isolation streaks were made on Mannitol Salt Agar (MSA) plates. MSA plates inhibit the growth of Gram negative bacteria, thus isolating the Gram positive bacteria. Once isolated, incubated, and grown, Gram stains were performed. Gram stains determined the Gram negative bacteria shapes were rods and Gram positive were cocci. Once Gram positive cocci were discovered, a Nitrate test was performed to determine which Gram positive cocci the bacteria could be. The Nitrate test narrowed the Gram positive cocci to Staphylococcus aureus. Two gelatin tubes were then made to determine which Gram negative rod. Gelatin tubes were incubated for 48 hours, and then refrigerated for a few days. Meanwhile, Glycerol and Galactose tests were also run on the Gram negative rod. These test results determined that the Gram negative rod was Pseudomonas aeruginosa.
The following tests were performed on this unknown:
1. Mannitol Salt Agar
2. Nitrate Test
3. Gelatin Test
4. Galactose Test
5. Glycerol Test
RESULTS
Flow Chart
Unknown #122
Gram negative rod
Gelatin test (Positive)
Positive Negative
Pseudomonas aeruginosa Escherichia coli
Klebsiella pneumonia
Enterobacter aerogenes
Proteus vulgaris
Galactose Test (Negative)
Positive Negative
Pseudomonas aeruginosa
Glycerol Test (Negative)
Positive Negative
Pseudomonas aeruginosa
Unknown #122 Gram negative – Pseudomonas aeruginosa
Flow Chart
Unknown #122
Gram positive cocci
Mannitol Salt Agar (Positive)
Positive Negative
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis
Nitrate Test (Positive)
Positive Negative
Staphylococcus aureus Enterococcus faecalis
Unknown #122 Gram Positive- Staphylococcus aureus
DISCUSSION/CONCLUSION
The Gram-negative bacterium was found to be Pseudomonas aeruginosa. The Gram stain for this bacterium showed a Gram-negative rod. It was then isolated and grown on an EMB plate. Further tests including gelatin (positive for hydrolyzing the gelatin), galactose (negative result), and glycerol (negative result) tests were performed to determine which Gram-negative rod the bacteria was. All of these tests worked well and didn’t encounter any problems.
Pseudomonas aeruginosa is a respiratory and skin pathogen that produces elastase and collagenase, which digest elastin and collagen (proteins found in connective tissue) (2). P. aeruginosa is found in water, soil, plants, and skin flora. This Gram negative rod is aerobic, meaning it needs oxygen to thrive. It is becoming increasingly recognized as an opportunistic pathogen of clinical relevance. It rarely infects healthy tissue, however when compromised, it can affect almost any tissue. Its been known to cause Urinary tract infections, respiratory infections, bacteremia, dermatitis, bone and joint infections, and a variety of other systemic infections (especially in cancer, burn, or AIDS patients).
It is also resistant to many commonly used antibiotics (3). This bacterium is naturally resistant to many antibiotics due to its tendency to colonize surfaces in the form of biofilms, which makes the cells impervious to therapeutic concentrations of antibiotics. Also, since its natural habitat is soil, P. aeruginosa is used to living in association with bacilli, actinomycetes, and molds, therefore it has developed a resistance to many naturally occurring antibiotics (3).
Staphylococcus aureus was found to be the Gram-positive cocci in unknown #122. Using a MSA plate isolated the bacterium and a Gram stain was done, which showed purple clusters. Two tests were then performed to determine this result. These include the MSA plate, which had a positive result for producing acid (agar turned yellow), and a Nitrate test, which also had a positive result (broth remained colorless). As with the Gram negative bacteria, the test results for the Gram positive had no problems and worked well.
REFERENCES
1. McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General Microbiology. Apr. 2011.
2. Park Talaro, Kathleen, and Barry Chess. Foundations in Microbiology. Eighth ed. Boston: McGraw-Hill, 2012. Print.
3. Todar, Kenneth, PhD. “Pseudomonas.” Todar’s Online Textbook of Bacteriology. N.p., 2012. Web. 03 Dec. 2012.
http://textbookofbacteriology.net/pseudomonas.html.
