Results: In order to identify the gram negative isolate in the unknown #260 broth, I observed the bacterium under the microscope, created a stock culture, and conducted a variety of biochemical tests. First, I inoculated a blood agar plate and a MacConkey (MAC) agar plate with the unknown #260 mixed culture broth. The blood agar plate, shown in Figure 1, was inoculated using the quadrant streaking method and then incubated for 24 hours at 35 °C. In the third and fourth quadrants, two different bacterial cultures were present. The bacteria that covered most of the plate were light gray, circular, convex, entire colonies in a punctiform arrangement with slight hemolysis around the mixed sample in quadrant 1. The other bacteria were more sparse
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Since MacConkey agar is selective for gram-negative bacteria (1), I was able to compare the growths on this plate (Figure 2) to those on the blood agar plate (Figure 1) and matched the colorless, flat, circular, undulate colonies on the MAC plate with the very light gray, irregular, flat, undulate colonies on the blood agar plate and identified them as the gram negative isolate. Due to the disappearance of my MAC plate, I had to isolate the Gram negative bacteria from the blood agar plate. Since there were matching colonies on the blood and MAC plates, I was able to identify the gram negative colonies from the gram positive growths and create a gram stain to confirm this identity. The result of the gram stain, shown in Figure 3, consisted solely of pink bacteria, which confirmed this was the gram negative isolate in accordance with Hans Christian Gram, the developer of the gram stain, who stated pink bacteria were gram negative (2). Since there was only one type of bacteria present on the gram stain slide, I confirmed that the sample was uncontaminated and used the colonies to make a stock culture (Figure 4). The MacConkey agar also differentiates between lactose fermenters and non-lactose fermenters, with lactose fermenters causing a color change of the medium from red to bright pink (1). Since the medium changed to a yellow color (Figure 2), I confirmed that the gram negative isolate had a negative result on the lactose test. Using this result, the unknown separation outline directed me to perform an Indole test. From the results shown in Figure 5, the lack of black precipitate indicates a negative H2S test and the lack of a red ring formed at the top of the tube after adding Kovac’s reagent indicates a negative Indole test (3). However, the bacteria did grow beyond the initial stab line, which indicates that the bacteria is motile (3).
Since MacConkey agar is selective for gram-negative bacteria (1), I was able to compare the growths on this plate (Figure 2) to those on the blood agar plate (Figure 1) and matched the colorless, flat, circular, undulate colonies on the MAC plate with the very light gray, irregular, flat, undulate colonies on the blood agar plate and identified them as the gram negative isolate. Due to the disappearance of my MAC plate, I had to isolate the Gram negative bacteria from the blood agar plate. Since there were matching colonies on the blood and MAC plates, I was able to identify the gram negative colonies from the gram positive growths and create a gram stain to confirm this identity. The result of the gram stain, shown in Figure 3, consisted solely of pink bacteria, which confirmed this was the gram negative isolate in accordance with Hans Christian Gram, the developer of the gram stain, who stated pink bacteria were gram negative (2). Since there was only one type of bacteria present on the gram stain slide, I confirmed that the sample was uncontaminated and used the colonies to make a stock culture (Figure 4). The MacConkey agar also differentiates between lactose fermenters and non-lactose fermenters, with lactose fermenters causing a color change of the medium from red to bright pink (1). Since the medium changed to a yellow color (Figure 2), I confirmed that the gram negative isolate had a negative result on the lactose test. Using this result, the unknown separation outline directed me to perform an Indole test. From the results shown in Figure 5, the lack of black precipitate indicates a negative H2S test and the lack of a red ring formed at the top of the tube after adding Kovac’s reagent indicates a negative Indole test (3). However, the bacteria did grow beyond the initial stab line, which indicates that the bacteria is motile (3).