In this lab experiment we did several test to determine what our unknown bacteria was. To determine this we recorded the results of how the bacteria reacted to different media. Depending on the results of each test we could narrow down the different bacteria to determine what our unknown is. This experiment will also determine if our bacteria is a fermenter of sugars and if it is catalase positive. If the bacteria is a fermenter they will use the sugars to make ATP. If the bacteria is a fermenter of lactose/sucrose the EMB plate we used will “clearly differentiate between the colonies of lactose fermenting and non-fermenting microbes. In the same medium sucrose was also included to differentiate between coliforms that were able to ferment sucrose more rapidly than those that were unable to ferment sucrose” (Cheeptham & Lal, 2007). If it is catalase positive it will produce bubbles with the drops of hydrogen peroxide on the bacteria. This will show if the bacteria is an aerobic or aero tolerant anaerobes or true anaerobes. Catalase is an enzyme that will take hydrogen peroxide and break it down to water and oxygen.(Merriam Webster 2013) “Catalase is thought to be a major defense against hydrogen peroxide” (Eaton & Ma, 1992). We will also determine if the bacteria is stable or non-stable acid fermenters. If it is stable it will produce the acid and it will be a positive result. This test will tell us the pathway of fermentation. Another thing we will determine is that if there will be a presence of citrate permease. Which will increase the ph level. (Health and Life Sciences Department 2013) By doing these tests we will be able to see how the unknown bacteria reacts to certain conditions. If our results of these lab experiments are conclusive then we should be able to determine what our unknown bacteria is.
Materials and Methods
Tube with unknown bacteria in a broth
Bunsen burner and Striker Inoculating loop
Tape for labeling tubes and plates
Test tube rack
Nutrient Agar Plate
Tube of MR-VP (methyl red)
Tube of Simmons citrate
We started our experiment by inoculating a tube of Methyl Red (MR-VP) with our unknown bacteria using the aseptic technique and our loop by swirling it in the media. We capped the tube and we then incubated the tube at 37 degrees Celsius for five days then refrigerated until the next class. We then added five drops of methyl red and gently tapping the tube to distribute. Next we inoculated a Simmons Citrate agar slant using the needle in the aseptic technique. We inserted the needle in the unknown bacteria and then we inserted the needle into the agar to the bottom of the tube and pulled it up along the slope of the slant. We then capped the tube and inoculated the tube at 37 degrees Celsius in an incubator for seven days then refrigerated until the next class. Our third test was on a nutrient agar (NA) plate. Again using the aseptic technique we streaked our unknown bacteria down the center of the plate in a heavy line using the loop. This plate was sealed with para film, flipped upside down and incubated at 37 degrees Celsius for 16-24 hours then refrigerated. At our next class we added three drops of hydrogen peroxide using the dropper directly to the bacteria line to test for catalase. We ended our experiment using the Eosin Methylene Blue (EMB) plate. Using the aseptic technique we streaked the plate with the unknown bacteria with the loop in a T-streak method. The plate was sealed with para film, stored upside down and incubated at 37 degrees Celsius for 24 hours then refrigerated
In the MR-VP test we did with the unknown bacteria we found that after adding the methyl red to the MR-VP Broth it turned red. By the color...
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