Two organisms, S. aureus and K. pneumoniae, were successfully isolated and identified from an unknown liquid culture labeled 11. These two organisms were identified through a series of tests and the process of elimination based on the flow charts I made for each organism. The tests chosen helped determine if the organisms could utilize a particular sugar, if the organisms contained certain enzymes and allowed for the differentiation between enteric organisms.
The purpose of this project was to use the techniques we have learned throughout this course to identify two unknown organisms that were originally taken from a liquid culture. Additionally, using the results from the various tests conducted allows for the opportunity to write a scientific paper on our findings. In the following paragraphs in this section, I will write about each organism I have successfully identified.
S. aureus is classified as a gram-positive, cocci, and a facultative anaerobe, which means it can grow in the absence or presence of oxygen by adapting to the changes in the environment (Masalha, Borovok, Schreiber, Aharonowitz, & Cohen, 2001). This organism is an opportunistic human pathogen, which means it does not cause infections until the host cell has a weakened immune system (Coates, Moran & Horsburgh, 2014). Typically S. aureus, most commonly MRSA, affects the skin and soft-tissue by causing terrible infections (King et al., 2006). S. aureus likes to bind to the skin to secrete enzymes that ruin the primary function of the skin, antimicrobial barrier (Coates, Moran & Horsburgh, 2014).
The primary place one can find S. aureus is in the nose and approximately 60% of the population are intermittent carriers. One common infection acquired in the hospital is nosocomial methicillin-resistant Staphylococcus aureus, otherwise known as MRSA, which is one strain of S. aureus. Patients who underwent a surgical procedure and/or have been catheterized have a higher chance of contracting MRSA. Infections caused by S. aureus have become difficult to treat due to the strains becoming resistant to multiple drugs (Foster, 2004).
K. pneumoniae is an opportunistic pathogen, which means it only causes a disease when the host cell has an impaired immune system. This organism does not have a high enough virulence factor to cause damage to a host cell with a normal immune system (Casadevall & Pirofski, 1999). Due to this organism being an opportunistic pathogen, it is often responsible for nosocomial infections, which frequently occur in the urinary and respiratory tract. K. pneumoniae is also found in the gastrointestinal tract as it serves as a great place for bacteria to spread to other areas (Struve & Krogfelt, 2004).
Two characteristics of organisms that are common to nosocomial infections include gram-negative cells and a bacillus morphology. Other hospital-acquired infections gram-negative bacilli typically cause include infections at the surgical site and in the bloodstream, as well as pneumonia (Weinstein, Gaynes, Edwards, & National Nosocomial Infections Surveillance System, 2005). This is not unexpected because it was mentioned earlier K. pneumoniae cause infections when the immune system is weak and when individuals go to the hospital they typically have a weakened immune system.
In addition to its presence in the human body and its tracts, this organism can also be found in animals, sewage, and polluted waters and soils (Bagley, 2015). Not only is K. pneumoniae gram-negative and rod-shaped, it is also a non-motile and facultative bacterium belonging to the Enterobacteriaceae family (Guo et al., 2012).
The organisms from the liquid culture were streaked onto a plate, which contained starch media, and was incubated. The following week the organisms were differentiated between based on shape and size, which allowed for each organism to be streaked onto two separate plates. Bacterium 1 was cultured using the T-streak technique and bacterium 2 was cultured using the Quadrant streak technique. These two plates were the incubated for a week and the cultures were used to carry out a variety of tests to help identify which two species were present in the liquid culture.
Once the two species of bacteria were isolated onto two different plates, a gram stain was completed to identify the morphologies of each organism and determine if the bacteria were gram-positive or gram-negative. With both of the organisms, a bacterial smear was completed, which allowed for the bacteria to be stained with crystal violet, Gram’s iodine, rinsed with water and a decolorizer, and stained again a counter stain, safranin. The bacteria stained purple have gram-positive cells, whereas the bacteria stained pink have gram-negative cells.
In the casein hydrolysis test, an inoculating loop is used to transfer bacterium 1 from the T-streak plate to a different plate containing agar and skim milk, which is used as a supplement for the casein source. The plate is incubated at 37°C overnight and the plate is then observed for growth and a zone of clearing. A positive result is indicated by growth and the presence of a zone of clearing, which means the organism produces casease. Casease, an enzyme, is used to degrade casein so it can cross the cell membrane and the organism can now use it. A negative result shows no growth or a zone of clearing, which means this organism does not produce the enzyme casease.
The urease test was conducted second on bacterium 1 and last on bacterium 2. The oranisms were taken from the T-streak plate and the Quadrant streak plate, respectively, and streaked onto slants in two different tubes. The tubes were then incubated for 24-48 hours at 37°C. A positive test is indicated by the media turning pink, which means urease is released to hydrolyze urea to ammonia and carbon dioxide. A negative test is indicated by media turning orange, which means the bacterium could not release the urease enzyme to convert urea to ammonia and carbon dioxide.
The catalase test was conducted next on bacterium 1. On a clean glass slide, one drop of
hydrogen peroxide was added and then cells from the T-streak plate were smeared onto the hydrogen peroxide drop. If bubbles were observed, this indicated the organism contains catalase and that catalase is during the hydrogen peroxide dropped onto the glass slide into water and oxygen. The oxygen being produced is what can be seen as the bubbles. If bubbles were not observed, the organism did not contain catalase and hydrogen peroxide was not turned into water and oxygen.
The sucrose test was the first test conducted on bacterium 2. One tube, containing the sucrose sugar, was taken and was inoculated with bacterium from the Quadrant-streak plate culture. The tube was incubated for 37°C for 24-48 hours and changes in color were observed. A red color indicated no fermentation occurred, an orange color indicated fermentation was occurring at the time, and a yellow color indicated fermentation occurred, such that the solution became more acidic and CO2 was produced.
The SIMS test, which was the second test conducted on bacterium 2, tests for three different variables including: production of sulfur, production of indole, and motility. The media in a SIMs tube includes casein, animal proteins, sodium thiosulfate, and ferrous ammonium sulfate. An inoculating needle was used to pick a small amount of cells from the Quadrant-streak plate culture and stab those cells into the center of the SIMs tube. The tube was then incubated at 37°C for 24 hours. After the incubation period, the tube was checked to see if the bacterium was motile, produced indole, and if it produced H2S.
If the organism did not stray from the line they were stabbed in, then they were non-motile. If the organism did stray from the line, they are considered to be motile. Finally, 5 drops of Kovacs Reagent were added to test for the production of indole and H2S. If a red ring is present at the top of the media, then the cells of the respective organism are positive for indole. If the red ring is not present, then the cells are negative for indole. If the media turned black, the cells produced H2S, whereas, if the media did not turn black, then no H2S was produced.
After determining this bacterium was gram-positive and had a coccus morphology, I decided to use the casein hydrolysis test first. If the results of this test were positive, I would have found my bacterium, but if negative I would have narrowed down the options to three different organisms. The next test I conducted was then urease because if I got a positive result I would know which species bacterium 1 was right away, but if not then it would narrow my options down to two. The last test I chose for this bacterium is catalase because of the quick results that can be obtained. The results obtained showed bacterium 1 was S. aureus.
After determining this bacterium was gram-negative and had a bacillus morphology, I decided to use the sucrose fermentation test because the results would inform me if this organism could utilize sugar or not, in this case it did utilize sugar, and would cut down the possible options of this organism to four. The next test I decided to use was the SIMs test because based on any of the results I could narrow down the possible options of my organisms to either one or two. The results of the SIMs test told me this bacterium was not motile, unable to produce H2S or indole, which narrowed me down to just two options. The next test I decided to run was urease because it would allow me to differentiate between the two remaining organisms, P. aeruginosa and A. faecalis. Based on the positive result, a bright pink color, told me the second bacterium from my liquid culture was K. pneumoniae.
Based on the results of the various tests conducted, bacterium 1 was found to be S. aureus. According to an article written by Masalha, Borovok, Schreiber, Aharonowitz, & Cohen in 2001, they stated S. aureus is a gram-positive organism with a cocci morphology and a facultative anaerobe. The gram stain I conducted confirmed this organism was gram-positive and had a cocci morphology. The positive results from the catalase test infers this organism is tolerant to oxygen because it contains the enzyme catalase, which converts hydrogen peroxide to oxygen and water.
The casein and urease tests were used primarily for the process of elimination, which helped me determine bacterium 1 was S. aureus. The two negative results indicated S. aureus did not contain the enzymes casease and urease. One strange and unexpected result was that the urease test conducted turned out to be false positive. Many individuals who either had S. aureus or S. epidermidis had troubles with the urease test. Therefore, the false positive test could be due to the original master cultures being switched or that they were contaminating one another.
If I were to do this project again, I would have conducted an oxidase test to confirm this organism was indeed a facultative organism. To conduct an oxidase test, a wooden stick is used to smear some cells from the plate culture onto the filter paper. Once the cells are smeared onto the paper, one to three drops of oxidase reagent is added. If the color turns to beige or cream, it is an oxidase negative organism and if the color turns to pink it is an oxidase positive organism. It is expected the test would have turned out negative, which means the organism does not have cytochrome c oxidase to oxidize the reagent. Therefore, other oxidases are being used in the electron transport chain.
Based on the results of the various tests conducted, bacterium 2 was found to be K. pneumoniae. This organism is known to be gram-negative with a bacillus morphology (Weinstein, Gaynes, Edwards, & National Nosocomial Infections Surveillance System, 2005). The gram stain test conducted confirmed these two characteristics of K. pneumoniae to be true. The positive result from the sucrose test confirmed this organism uses sucrose as a carbon source, which contribute to this organism’s pathogenesis (Gering & Brückner, 1996).
Based on the literature published by Guo et al. in 2012, K. pneumoniae is a nonmotile organism, which was confirmed by the results from the SIMs test. This test also showed this organism is unable to produce indole or H2S. This test is used when wanting to differentiate between the species in the Enterobacteriaceae family. The positive urease test confirmed bacterium 2 was K. pneumoniae, which was not unexpected because many enteric organisms are able to hydrolyze urea due to the presence of urease.