The purpose of this laboratory exercise was to perform tests necessary to be able to distinguish one microorganism from 10 others. Using a series of biochemical tests and characteristics, unknown #22 was concluded to be Pseudomonas aeruginosa. A dichotomous key was mapped out and used during this process. Using this provided guidance as well as organization as to what the result may be. Upon obtaining the unknown organism, it was important to make a streak plate of the bacteria on TSA. The purpose of doing so ensures that we have pure cultures of the unknown to be used in further testing and not a mixed culture. The first test used was a gram stain. It is a differential stain that helps distinguish between gram-positive and gram-negative bacteria. After performing the gram stain, it was clear that the unknown was a gram negative due to its pink color. Gram staining involves doing a simple smear, drying, and then heat fixing. Then using the staining technique with crystal violet, gram’s iodine, ethanol, and safranin, a pink or purple color should result when looking at the slide under a microscope. A gram-positive, or purple stained bacteria, means that there are multiple layers of peptidoglycan in the cell wall. A gram-negative means that there in a thin layer of peptidoglycan that is removed by the ethanol and stained pink by the counterstain. All gram-positive bacteria could now be ruled out. The gram-positive bacteria included: Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, and Staphylococcus epidermidis. Confirming that the unknown was a gram-negative bacterium, the next step on the flow chart was a glucose test. In order to do so, fermentation tubes were used. Fermentation tubes include peptone, an acid-base indicator (or phenol red), an inverted tube to see if there was gas production, and around 1% of the desired carbohydrate. After inoculating the glucose fermentation tube and incubating it, the color of the phenol red did not change. That means it was a negative for glucose fermentation. Negative or “K” means it is alkaline and there was no fermentation. If the tube turned yellow, but had no gas in the inverted tube (Durham tube), that would mean its result was “A” or acid. If the tube turned yellow and contained a bubble it would be “Ag” which codes for acid and gas. The next step would be to perform a urease test. Urea is a byproduct given off from protein digestion and is given off in urine. When the enzyme urease is present, it can help distinguish between bacteria. For this test, a urea agar is used that contains peptone, glucose, urea, and phenol red. The pH of the agar is 6.8 and is originally yellow. If the bacteria is positive for urease, ammonia will be produced and raise the pH of the agar resulting in a hot pink color. If it is negative, there will be no change from the yellow. After inoculating s urea slant plate and incubating it, the results turned out to be positive. The organism had indeed produced ammonia and turned the agar to a fuchsia color. Knowing that unknown organism #22 was a gram-negative, K for glucose fermentation, and positive for urease, it was concluded that the organism was Pseudomonas aeruginosa. Even though the unknown was identified after three tests, other tests were performed in order to confirm that it actually was. Other tests performed included H2S, lactose, catalase, citrate, starch hydrolysis, indole, and sucrose. Some bacteria have the enzyme desulfurase that removes hydrogen sulfide (H2S) from the amino acids cysteine and methionine. This requires inoculating a SIM (Sulfide-Indole-Motility) test tube and incubating it. It will test for desulfurase, indole, and motility all in one tube. After letting it incubate, if there is a black precipitate where the inoculating needle was stabbed in the agar, it is positive for hydrogen sulfide. Black precipitate is formed due to ferrous ion (Fe2+) in the medium that reacts with the sulfide (S2-) to produce precipitate. Results to the hydrogen sulfide test are variable and may be positive or negative for P.aeruginosa. There was also motility because there was growth away from the point of inoculation. It looked like an upside down Christmas tree in the tube. The last thing SIM tested for was indole production. It is produced when the bacteria converts amino acid tryptophan to indole. 5 drops of dimethylaminobenzaldehyde, or Kovacs reagent, was added to the test tube. Upon addition, if it is positive for indole, it will turn a red color at the top of the medium. When the unknown was tested, it did not produce a red color. There was a slight dark red/brown color, but overall the test result was negative. Lactose and sucrose tests were done, which are very similar to the glucose test. The only difference is the 1% carbohydrate in the medium. For sucrose, the medium contained sucrose. For lactose, the medium contained lactose. Both of the test tubes resulted in a K result. There was no color change to yellow or any gas produced in the inverted test tube. The color did change to an unusual bright pink, but it did not turn yellow like it would have in the presence of acid. The unknown was also tested for catalase. Aerobic bacteria produce the enzyme catalase. It breaks downs hydrogen peroxide into water and oxygen. After smearing an isolated colony of the unknown on a slide, hydrogen peroxide was added and it resulted in bubbling. Bubbling suggests a positive test and if there is no bubbling, it is a negative test. A citrate lyase test was also done using citrate agar. Bacteria with this enzyme break down citrate to form pyruvate. Simmons citrate agar is used. When bacteria use citrate and ammonium, the medium becomes basic because ammonia is produced. If positive, the media will turn from a green color to blue. After inoculating the Simmons citrate agar and incubating, the media turned to a blue color. The unknown was positive for citrate lyase. Lastly, as a reconfirming test, a starch hydrolysis test was done. Exoenzymes are mainly hydrolytic enzymes. These enzymes leave the cell and break down with the addition of water. Amylase then hydrolyzes the polysaccharide starch into smaller components. Glucose is release and there is usually a substrate outside of the bacteria colony. Using a starch agar, the media was inoculated and incubated. After incubation, iodine was added to the plate. A clear outer ring of where the media was inoculated means that it is positive for starch hydrolysis. When doing this with the unknown, it resulted in a negative starch hydrolysis test. After all these test results, it was safe to say that unknown #22 is Pseudomonas aeruginosa. Using the dichotomous key, the bacteria had already been identified after three tests, but it is important to do additional confirmation tests as well. Not all of the tests gave straightforward answers but with the chart given and dichotomous key, it was possible to confirm using other tests. The tests used were: glucose fermentation, sucrose fermentation, lactose fermentation, indole, catalase, citrate, urease, starch hydrolysis, and H2S. These tests helped confirm that the unknown was P. aeruginosa.