All is the cell wall, which maintains cell

All organisms are divided into three domains; bacteria, archaea, and eukarya. The organisms making up domain Bacteria and domain Archaea are all prokaryotes. Although bacteria and archaea look the same, archaea is more closely related to eukarya (Madigan 2009). The ability to adapt to a broad range of habitats helps to explain why prokaryotes are the most abundant organism on earth. The main characteristics of a prokaryote include, no nucleus, circular DNA, and no membrane bound organelles.A key feature of nearly all prokaryotic cells is the cell wall, which maintains cell shape, and provides

physical protection. Most bacterial cell walls contain peptidoglycan, a network of modified-sugar polymers cross-linked by short polypeptides. All known pathogenic bacteria fall under prokaryotes, but not all bacteria are pathogenic (Madigan 2009). Using a differential staining technique bacteria can be divided into two groups; gram positive or gram negative. The gram-positive bacteria have thick cell wall made of peptidoglycan. Gram-negative bacteria have thinner layer of peptidoglycan, and are structurally more complex, with an outer membrane that contains lipopolysaccharides. Gram staining is a valuable tool to determine whether an unknown bacterium is gram negative or gram positive. Samples are first stained with crystal dye and iodine, then rinse in alcohol, and finally counterstain with safranin. In gram-positive bacteria the crystal violet adheres to the peptidoglycan layer staining it purple,. In gram-negative the crystal violet is easily removed due to the thinner layer of peptidoglycan making the cell appear pink or red. (Leboffe 2010). The domain bacteria is very diverse, making it impossible to identify it without the use of staining and biochemical tests. Biochemical tests can test for a variety of characteristics such as shape, morphology, pH, etc. that are specific to a certain type of bacteria. Tests such as Methyl Red and Voges- Proskauer check for glucose fermentation. The Citrate Test is a nutrient utilization test that determines whether a bacterium produces the enzyme citrate- permease to be utilized as its sole source of carbon. Urease test is used to indicate if a bacterium can hydrolyze the intracellular enzyme urea. The Gelatinase test indicates

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whether or not a bacterium contains this exoenzyme which hydrolyzes the proteins and allows the gelatin to be converted into solid when is chilled. Two types of combination differential media biochemical tests were used; SIM (Sulfur Indole Motility) and TSIA (Triple Sugar Iron Agar). SIM gives three test results; Sulfur reduction, which determines whether a bacterium can use enzymes to reduce sulfur to hydrogen sulfide, Indole production used to determine if the bacteria can hydrolyze tryptophan to pyruvate, ammonia, and motility when in the semisolid media a motile bacteria can diverge from the stab line. TSIA is designed to differentiate bacteria with reference to glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur reduction (Leboffe 2010). Materials and Method To prevent errors in any procedure personal protective and equipment aseptic technique was strictly used. These techniques included sterilizing the loop on the Bunsen burner between inoculations and flaming the opening of the test tube before inserting in the loop with the bacteria. To isolate pure single colonies the unknown # 31 microorganism was T-streaked on a TSA plate and a TSA slant for backup. The plate was incubated for 24 hours in a in a 37 degree Celsius room and then transferred to a cold room 4degree Celsius room. After incubation, a gram stain was performed on the isolated single colonies to determine if the unknown microorganism was indeed gram negative. First, the organism was smeared onto a slide with a drop of distilled water. The

smear was allowed to air dry and heat fixed right after to provide bacteria to stick onto the slide. Staining started with crystal violet, which is used to flood the slide completely for a minute and once again rinsed for about 10 seconds with DI water. The slide was blotted dry with bibulous paper and was observed using the microscope under 100x oil immersion. The color, shape, and morphology of the bacteria were recorded. The first and easiest test conducted was an oxidase test, which is use to test for the presence of cyrochrome c oxidase. A small amount of the culture was transferred onto a reagent paper, and rinsed with a drop of DI water. Data was recorded within 20 seconds. Next the TSIA test was performed. The test proves whether a bacterium can ferment carbohydrate, produce gas and produce Hydrogen sulfide. The bacterium was swiped on to the needle and was stab two thirds down the TSIA media then streaked along on the TSIA slant. It was later placed in the hot room for 24 hours. After 24 hours the TSIA and SIM test medium were taken out to observe the results. For the TSIA, the butt and slant were inspected for color change. Yellow indicates positive for glucose and lactose fermentation with acid accumulation, and black precipitate (sulfur reduction), and gas production. The SIM test was performed to test for sugar fermentation, indole and motility. The semisolid medium contains casein, amino acids, an iron-containing compound, and sulfur in the form of sodium thiosulfate. The SIM tube was first inspected for back precipitate indicating sulfur reduction and cloudiness around stab line indicating motility. Kovacs reagent was added in the medium (a depth of


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