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Bacteria are basically everywhere; they are a part of our environment and even in us. Therefore, we come in contact with bacteria everywhere and it is sometimes necessary to identify them. Whether it is to determine the cause of a disease, to test if a certain food is safe to eat or simply to know what is present in a certain ecosystem, we have industrialized many methods to identify certain types of bacteria. Every species is distinctive and has particular characteristics that go along with it. This makes it promising to classify an unidentified species. The appropriate identification of a microorganism is not only significant in a microbiology lab but also in industrial, medical, and pharmaceutical fields. (Lloyd et al. 2003)When an unknown bacterium is found, a pure culture of it on an agar plate must be made first. A pure culture arises from a single cell and thus contains only one type of microorganism. A colony is a visible mass of cells. Each bacterial species creates different culture morphologies. The process of identifying unknown bacteria usually starts out by noting their physical characteristics, such as cell wall, and shape. Using standard laboratory procedures, like cell staining, and culturing we can further narrow down the identification. Microorganisms are usually arranged into species according to their physical and metabolic features (Janda et al. 2002). Bacteria with thick cell walls are termed gram-positive because they’re prone to dying following the addition of crystal violet during the gram stain. The gram stain is the first test used in bacterial classification. Bacteria with thin or absent cell walls, termed gram-negative because they do not retain cystal violet in their membranes following the addition of alcohol. Differential and special stains are also needed to reveal characteristics like: gram-negative and gram-positive bacteria, Acid fast and non-acid fast, spirochetes, capsule and Flagella, etc (Lupetti et al. 2010, Lupetti et al. 2013)Lab procedures learned during this course were executed to separate and identify two unknown microorganisms. The purpose of the unknown lab is to apply the systematic reasoning of a microbiologist to properly identify the species of two prokaryotic bacteria from one unknown sample. MATERIALS/METHODSThe tests performed throughout the course of identifying unknown samples from nutrient agar plate l were followed exactly as detailed in the text, Microbiology: The Laboratory Experience (Keating 2016). The significance of this method is to isolate pure colonies of bacteria and be able to begin testing each organism separately. In order to obtain a pure culture of an organism, the isolated colonies are aseptically relocated to a different nutrient agar followed by utilizing the quadrant method with a wire and incubated overnight at 37 degree Celsius (Sanders 2012). to detect presence of catalase DISCUSSIONThe first microbe unknown was classified to be a gram-positive bacillus shaped, Mycobacterium smegmatis. After the isolation of both organisms by plating on nutrient agar and selecting isolated colonies, I confirmed that the isolation was successful by analysis of their physical appearances.  I observed the overall appearance of growth to be rough, elevation was convex, form was irregular, and margins were undulated, all characteristic of M. smegmatis.  I initially differentiated that the organism was gram positive following the streaking of the organism on MacConkey agar and a bile esculin slant. The results of no growth on the MacConkey and the bile esculin differential media confirmed my hypothesis since these media are designed to selectively inhibit gram-positive organisms. Negative lactose and sucrose fermentation test results removed Micrococcus luteus as a possibility and further confirmed the presence of M. smegmatis. Streaking on 5% sheep blood agar indicated gamma-hemolysis (no hemolysis of red blood cells) because there were no notable clear zones around the colonies that had formed. This result removed Staphylococcus aureus, Streptococcus pyogenes and Streptococcus agalactiae, that are all b-hemolytic and Streptococcus pneumonia and Streptococcus mitis that are a-hemolytic. A negative citrate result agrees with the negative outcome of the test done on M. smegmatis by Public Health England (reference?).  The following tests were performed to further confirm the presence of M. smegmatis. Negative results from the mannitol, indole tests all paralleled the metabolism of M. smegmatis. In order to confidently conclude the gram-negative organism was indeed Enterobacter aerogenes, I initially analyzed the colony morphology on selective media. When grown on MacConkey’s agar, pink/red mucoid colonies formed because it ferments lactose and produces acidic byproducts. On eosin methylene blue agar I observed pink colonies with a purple dot in the center of the colonies, which is specific to E. aerogenes. These physical characteristics removed Klebsiella pneumoniae as a potential option. The following series of tests performed on the unknown led to E. aerogenes as the only possible candidate. A positive result on MacConkey’s agar and the lactose broth eliminated Salmonella spp, Shigella spp., Proteus spp., Pseudomonas aeruginosa, and Serratia marcescens as possibilities since they cannot ferment lactose. A positive citrate test removed Escherichia coli as a possibility. A negative oxidase test removed Alcaligenes faecalis as a possibility. I performed the following biochemical tests to further verify the presence of E. aerogenes as my microbial unknown. A positive mannitol test, esculin test, and catalase test all corresponded with E. aerogenes metabolism. I was able to identify two unknown microbial species by first, isolating and noting their morphological appearances and performing various biochemical tests. In light of recent technological advances, such as genetic testing, this approach is not specific enough considering that certain species can have variable outcomes to a variety of the tests that were performed. Molecular techniques such as DNA sequencing, which is currently the method of choice for identification of unknown bacteria is highly specific and useful for classifying bacteria by species (Fraser et al. 2000). In future lab experiments, if the cost will not be too high, I would recommend that students could sequence their unknown to narrow down their identification. 

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