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Tests for How to Identify P. aeruginosa | Microbiology Unknown Lab

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Unknown #119

Molly Doyle
Professor Snaric
Microbiology – BIO 203





Bacteria play a huge role in our lives every day. The word “bacteria” may carry a negative connotation with the general public; however, there are many bacteria that help keep us alive and healthy. Both helpful and harmful, they are everywhere and we cannot avoid them, no matter how hard we try. Over the years, scientists have found a way to differentiate and prove if a particular bacterium is pathogenic (disease-causing) or not. This study utilized the tests that have been known to validate the presence of specific bacteria by microbiologists and students alike.


The lab instructor randomly assigned each student an unknown mixed bacterial specimen. The tests that were introduced and practiced throughout the semester were to be used. These procedures were followed according to the instructions listed in the lab manual (McDonald et al. 2011).

The first step taken was to streak plate the mixed culture onto a nutrient agar plate. Proper streak plating method is explained on page 10 of the lab manual (McDonald et al. 2011). This first step was critical in isolating the two bacteria present in the culture. The nutrient agar was put into the incubator and kept at 37 degrees Celsius.

Five days later, the nutrient agar showed insignificant growth, so the streak plate was repeated and placed in the 37-degree incubator (the original streak plate was thrown away). Two days later, the repeated streak plate showed a copious amount of growth. The growth only appeared to be one particular colony type, so a single colony was transferred to a new agar, in hopes that only one bacterium would be present. The agar was placed in the incubator again and kept at 37 degrees Celsius.

Five days after the bacterium was isolated, there appeared to be two distinct bacteria in what was thought to be an isolated plate. Fuzzy and light colonies were found, as well as smooth and dark colonies. The next test was a gram stain (McDonald et al 2011). After two unsuccessful attempts of the gram stain, the colonies were taken from the isolated plate and streaked on a brand new agar to start fresh.

Two days later, one of the instructors came into the lab to help and discovered that the mixed culture of bacteria would only show one type of colony growth and inhibit growth of the other unknown, due to its overpowering nature. Since the colonies had a green glow to them, they were deemed as Pseudomonas aeruginosa (P. aeruginosa). A second attempt at gram staining was performed and the bacterium was found to be gram-negative, bacillus shaped. To be sure of the presence of P. aeruginosa, a gelatin test was performed (McDonald et al. 2011). In order to identify the gram-positive bacterium, an alternate tube (#9) was given. This alternate bacterium was streaked onto a new nutrient agar and incubated at 37 degrees Celsius.

After another five-day period, the alternate bacterium showed considerable growth. A gram stain was performed on alternate #9 and was found to be gram-positive, bacillus shaped. In order to further determine which bacterium the alternate tube contained, two more tests were run. The Maltose and Methyl Red test would both confirm which gram-positive bacillus bacterium was present (McDonald et al. 2011).

The following tests were successfully implemented on Unknown #119/Alternate #9:
1. Gelatin
2. Maltose
3. Methyl Red

Table 1. Alternate #9 Test Results

Test Purpose Reagents Observations Results
Gram stain To determine gram reaction of the bacterium Crystal violet, Iodine, Alcohol, Safranin Purple rods Gram positive bacillus
Maltose To determine carbohydrate fermentation Maltose test tube Pink color Negative maltose test
Methyl Red To determine glucose fermentation Methyl red test tube Light orange color Negative methyl red test

Table 2. Unknown #119 Test Results

Test Purpose Reagents Observations Results
Gram stain To determine gram reaction of bacterium Crystal violet, Iodine, Alcohol, Safranin Red rods Gram negative bacillus
Gelatin To determine use of gelatin as carbon or energy source Gelatin test tube


Green ring at top of broth tube Positive gelatin test

Table 1 and 2 list the biochemical tests performed in this study, their purpose and the results. Results are also shown in flow chart form below.

The only test for the gram-negative bacterium was the gelatin test. A green ring was produced at the top of the test tube after being in 37-degree incubator for two days.

The unknown gram-positive bacterium required two tests. First, the maltose test was performed. After two days of incubation, the test tube medium turned from a deep red color to a lighter, pink color. Next, the methyl red test was allowed to incubate for two days and turned from yellow to an orange tint upon addition of 5 drops of the methyl red (.2%) solution.

FLOWCHART: Alternate #9


Gram stain


Gram-positive bacillus


Methyl Red (negative)

Positive                                                                                  Negative
Bacillus cereus                                                                       Bacillus subtilis
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis

 Maltose (negative)

                                                                        Bacillus cereus                       Bacillus subtilis
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis



FLOWCHART: Unknown #119

Gram stain


Gram-negative bacillus


Gelatin (positive)

Positive                                                                                  Negative
Pseudomonas aeruginosa                                                       Escherichia coli
Klebsiella pneumoniae
Enterobacter aerogenes
Proteus vulgaris

The test for the gram-negative bacterium was the gelatin test. This test was performed because the only gram-negative bacterium that would test positive for the gelatin test was the P. aeruginosa. The green ring that was produced at the top of the test tube demonstrated that the bacterium in question was in fact P. aeruginosa. This bacterium was the only one that would result in a positive gelatin test, confirming its identity.

The unknown gram-positive bacterium was found through two separate tests. First, the maltose test was performed. After incubation, the test tube turned from a deep red color to a lighter pink, demonstrating a negative maltose test. Next, the methyl red test tube turned from yellow to an orange tint, which was also deemed a negative test.

One major issue was encountered in this study. The P. aeruginosa outgrew the other unknown bacterium, inhibiting growth of anything else. Therefore, it was not possible to separate the two bacterial cultures on the streak plate. This issue was not identified for most of the study and was a major setback in the findings.

This stubborn bacterium, P. aeruginosa, has intriguing associations in a clinical setting. This bacterium can grow inside the human body without harming it, until it forms a biofilm, which is then able to overpower the immune system (Tortora et al. 2007). Typically, P. aeruginosa is commonly found in soil and water, and on the surface of plants and animals. It is also considered a nosocomial (originating in a hospital) pathogen, affecting any compromised tissue (Todar 2012). One reason that this bacterium is dangerous is due to its high tolerance to unfavorable environments. Since P. aeruginosa is so prevalent in the environment, it is likely that a person has been and will be exposed to the bacterium often. While exposure does not pose an immediate threat, P. aeruginosa is a common opportunistic pathogen (Tortora et al. 2007). This means that the bacterium can be the cause of a secondary infection, often fatal.

This bacterium has been found to be responsible for UTI’s, respiratory infections, dermatitis, bone/joint infections and gastrointestinal infections (Todar 2012). Patients who are immuno-suppressed are also at high-risk of being affected by P. aeruginosa. Those burdened by cancer, AIDs, or severe burns are especially susceptible due to the high levels of stress on the body. In a hospital, the immune system of a patient is almost always suppressed or compromised in some way. This allows a perfect opportunity for P. aeruginosa to be introduced into the body and take over. The optimal temperature for P. aeruginosa to grow is 37 degrees Celsius, which is body temperature. These factors make hospitals vulnerable to infections caused by P. aeruginosa.

This bacterium is known to be resistant to most common antibiotics, due to the R factor on their plasmids, which carries genes to be resistant to these antibiotics (Tortora et al. 2007). Antibiotic resistance is a serious issue in healthcare today. The durability of P. aeruginosa is just one of many culprits that are lurking in hospitals and clinical settings, waiting to overtake the immune system and eventually, render the body helpless.

Fortunately, new antibiotics have been formed to fight P. aeruginosa. Quinolones and antipseudomonal beta-lactam antibiotics are used in the form of chemotherapy to fight the detrimental effects that P. aeruginosa poses in humans (Tortora et al. 2007). Being able to identify this bacterium in a quick and effective manner is helpful in order to properly treat any infections caused by P. aeruginosa. Using simple methods of identifying bacteria, such as the ones in this study, proves to be a great tool to help prevent major infections and save many lives.


McDonald V, Thoele M, Salsgiver B, Gero S. 2011, LAB MANUAL for General Microbiology. St. Louis Community College.

Todar, Kenneth. Online Textbook of Bacteriology, 2012. Madison (WI).

Tortora G, Funke B, Case C. 2007, Microbiology: an introduction, ninth edition. San Francisco (CA): Pearson Education.


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