Pseudomonas aeruginosa Report | Microbiology Unknown Lab

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UNKNOWN LAB REPORT

Unknown Number 106

Megan McBride

April 29^th, 2014

Microbiology

Introduction:

There are microorganisms on every single thing in our world. These microorganisms range from deadly to vital for life and health to continue. Either way, all microorganisms must be sought out and studied by scientists, some taking the same path that was traveled in this study. The road to discovering both unknown bacteria given by the instructor was a rocky one; seemingly countless trials and errors later, a conclusion was reached for both.

Materials and Methods:

Firstly, a streak plate was conducted on a nutrient agar plate in an attempt to isolate the bacteria from one another. Upon observation of the streak plate, it was found that there were no perfectly isolated colonies. However, the plate did show one mossy green colored bacterium and another yellow opaque-colored bacterium. A second streak plate was inoculated on a nutrient agar, and to have some semblance of progress a small, mostly isolated yellow opaque colony from the first streak plate was grown on a nutrient agar to determine if it was in fact isolated or not.

The result from the colony taken from the first streak plate and grown on the nutrient agar appeared to be contaminated. The agar was tinted with the mossy green bacterium, while the colony it was taken from was a yellow opaque colony. The results from the second streak plate were positive and showed one perfectly isolated colony. A gram stain was performed on this colony and revealed red rods, a confirmed gram-negative result. This colony was grown on a milk agar plate to gram stain once again to reconfirm that it is in fact isolated and tests on the bacterium could continue. Due to the fact that the gram-negative bacterium was isolated a lawn was created on a mannitol salt agar plate from the original broth, which selects for gram-positive bacteria to grow.

The mannitol salt agar plate used to grow the gram-positive bacterium returned with one yellow opaque isolated colony. However, when the colony was gram-stained it revealed red rods, a confirmed gram-negative result, which was the opposite of what was hoped for. The plate must have been contaminated as gram-negative bacteria never should have proliferated on a mannitol salt agar plate.

The isolated colony taken from the second streak plate and grown on a milk agar plate became completely taken over by the mossy green bacterium. A gram stain test was performed and purple rods revealed a confirmed gram-positive result. This was also an undesirable result as the milk agar was grown from a colony that was originally gram stained gram-negative. After consultation with the professor, it was explained that the gram-negative bacterium never should have been attempted to grow on a milk agar plate. From that point, the entire process was recommenced. A fourth streak plate was inoculated on a nutrient agar from the original broth, and a lawn from the original broth was also created on a mannitol salt agar plate in order to only grow the gram-positive bacterium.

The fourth streak plate returned with no isolated colonies so a fifth and final streak plate was created. The mannitol salt agar pate returned with four perfectly isolated colonies. The gram stain from one of the more isolated colonies revealed purple coccyx, a confirmed gram-positive result, which narrowed the list of possibilities down to three bacteria. Next, a urea, maltose, and nitrate test were conducted to further narrow down the possibilities of bacteria. The instructor was once again consulted when the results were inconclusive, and an alternate for the gram-positive bacterium was presented. The instructor explained that the mannitol salt agar plate gram stain should have returned gram-positive rods and not coccyx, which narrowed the list down to the first two bacteria that were eliminated. From the alternate gram-positive tube a gram stain was performed which confirmed gram-positive rods, a casein test, a maltose test, and a methyl-red test were all inoculated. All of the tests were conclusive except for the casein test, which both bacterium should have tested positive for. An oxidase test was executed to double-check the results which concluded the gram positive bacteria confirmed as Bacillus cereus.

Due to the fact that the fifth streak plate was once again unsuccessful, an alternate for the gram negative bacterium was also provided. The gram stain for the alternate revealed red rods, a confirmed gram negative result. A Simmons citrate and SIM tube tests were executed to narrow down the list of possible gram negative bacterium. Finally, urea and casein tests were performed to determine which bacterium was present. The urea test returned concluding that the gram negative bacterium was Pseudomonas aeruginosa.

Discussion/Conclusion:

Several rounds of trial and error were experienced throughout the unknown discovery process. After thinking that I was near the end several times, there was always a test that did not come back as it was supposed to. Thankfully there were alternate tubes that were pre-isolated as that was the part that I struggled with the most. While my tests on the alternates did not always go as planned, they did lead me to success. By narrowing down the list of possibilities with several tests at one time and confirming the results by doing tests which only had one possibility, I was able to reach the correct conclusion for both. I encountered several problems with my streak plates and isolating the bacteria from each other, none of them being correct. I also had trouble getting the correct bacteria to grow on the correct plates. I suspect that I had a lot of contamination, and as I got further along with the process that my broth had been out and used so long that it was leading to incorrect test results.

Pseudomonas aeruginosa is a bacterium that can cause an infection in almost any part of the human body. The most serious infections caused by P. aeruginosa often nosocomial, or in those with compromised immune systems. (1) However, those with healthy immune systems can also develop an illness from the bacterium. The bacterium can be spread in hospitals from such a thing as simple as health care workers not washing their hands, or from a piece of equipment that was not properly cleaned. The origin of the pathogen is widespread inhabiting soil, water, plants, animals, and humans (2). P. aeruginosa is the most common pathogen isolated from patients whom have been hospitalized for longer than one week. The bacterium acts as an opportunistic pathogen and therefore rarely causes disease in healthy people. The bacterium also has minimal nutritional requirements and can tolerate a wide variety of physical conditions, adding to its pathogenicity (2). The bacterium can cause a wide variety of diseases including endocarditis, respiratory infections, septicemia, central nervous system infection, ear and eye infections, bone and joint infections, gastrointestinal infections, urinary tract infections, and skin and soft tissue infections (3). P. aeruginosa affects nearly every system in one’s body, which is why it is so incredibly dangerous, especially present in hospitals.

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