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November 14, 2018 CMB 201 Unknown Lab Report

Introduction The purpose of the experiments that were conducted was to identify a sample of unknown bacteria. This bacteria was used in multiple tests to determine specific qualities that each possible bacteria option might have. My unknown bacterium was Staphylococcus epidermidis. This bacterium was identified specifically by the gram reaction, cell morphology, catalase test, glucose fermentation, and nitrate reduction. The purpose of the gram reaction test was to determine if it was gram positive or gram negative. The purpose of the cell morphology test was to determine the shape of the bacteria. The purpose of the catalase test was to determine if the bacteria was aerobic or anaerobic. The purpose of the glucose fermentation was to determine if the unknown ferments carbohydrates and the waste it produces. The purpose of the nitrate reduction was to determine if the bacteria reduces nitrates to permit anaerobic growth when there is little oxygen.

Materials and Methods All experiments conducted to determine the unknown bacteria used aseptic technique to ensure no contamination and limit possibility of error. The first experiment used to isolate a bacterium was choosing a colony and making a smear. This was initially made on an agar plate and was grown until the next class period. It was then put into two tubes to ensure we would have enough of the unknown to conduct our experiments.

The gram stain experiment included the following. Cover smear in crystal violet for 60 seconds. Use squeeze bottle to wash water over the smear. Cover smear in Lugol’s iodine for 60 seconds. Rinse with squeeze bottle of water. Cover smear with 95% alcohol and let sit for 10 seconds, then rinse off with water. Repeat this step. The last step is to cover the smear with safranin for 30 seconds. Rinse with the water and gently dry it. The cell morphology test is used by obtaining an NA plate and observing three different types of colonies. Gram stain these colonies using directions in previous experiments. Choosing one that is hopefully gram positive. The catalase test needs an agar plate and hydrogen peroxide. The unknown bacteria is inoculated on the agar plate and left to sit for two days. Apply one drop of 3% hydrogen peroxide to the plate and observe for bubbles.The glucose fermentation experiment consisted of using a glucose Durham tube inoculated with the unknown bacteria. They were observed for bubbles in the inverted tube at the next class period. The nitrate reduction experiment requires a nitrate broth tube. The tube needs to be inoculated with the unknown by swirling the loop inside the broth and scrubbing it on the sides of the tube. By using the results of all of these experiments and following the flow chart provided by the instructor it was able to be determined my unknown bacteria was Staphylococcus epidermidis. A  ll of these experiments were sourced directly from the lab notebook and class powerpoints published by the instructor.

Results See attached documents.

Conclusion and Discussion The identity of my unknown was found through the series of tests above. The key experiments that led me to a conclusion was the gram reaction, cell morphology, catalase test, glucose fermentation, and nitrate reduction. These five tests were the determinants of S. epidermidis. T  he gram reaction was an experiment used to determine if the bacteria was gram positive, which would result in a purple stain, or gram negative, which would result in a pink stain. Gram negative bacteria has an extra lipopolysaccharide and protein layer that causes the pink stain to occur. My bacteria was gram positive. The next test was the cell morphology. This observes the shape of the bacteria. This is determined by looking under the microscope at the possible shapes, rods/bacilli and cocci. My bacteria was cocci shaped. The next experiment to determine my bacteria was the catalase test. This test used a dropper to administer hydrogen peroxide into the sample of the unknown. If it bubbled then the sample would be positive for production of catalase. My sample bubbled. The next test was the glucose fermentation and in this test the presence of bubble in the inverted tube was observed. The bubbles indicated fermentation had occurred and that gas would be a waste product of the experiment. The final test is the reduction of nitrates. If there are bubbles present the unknown is positive for the reduction of nitrate to gas. If there is no change the TA will add sulfanilic acid and dimethyl-alpha-naphthylamine. If the culture turns red the unknown is positive. My unknown sample was positive for nitrate reduction. Bergey’s Manual labels S. epidermidis  as a gram positive, cocci shaped bacteria. The manual uses the catalase test and the mannitol fermentation. The catalase test should have a positive outcome while the mannitol fermentation should have a negative outcome. The next test used is observing for yellow pigmented colonies. S. epidermidis  should not be a yellow

pigmented colony. The final test observes its reaction with novobiocin sensitivity and S. epidermidis  should be sensitive to it. Some interesting facts about S. epidermidis i s it has genes to help cope with extreme salt concentrations. S. epidermidis  produces a layer of slime which becomes a hydrophobic biofilm. The bacteria has a diameter of 0.5 to 1.5 micrometers. S. epidermidis c an live on dry surfaces for long periods of time.

References Nat Rev Microbiol. 2009 Aug; 7(8): 555–567. Retrieved  from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/ Bukhari M. September 27, 2004 Retrieved from : http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.ht ml Castenholz, Richard W., et al. Bergey's Manual of Systematic Bacteriology . Springer, 2001. SPERRY, JAY. MICROBIOLOGY: Laboratory Manual for Allied Health and General Microbiology. KENDALL HUNT, 2018....