Identification of a Unknown Bacterium

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Introduction

Although bacteria is microscopic in size, it is largely important in the healthcare field, environmental work, food preparation, as well as many other industries. In particular, it is essential that healthcare workers be able to identify the species of bacteria invading a human reservoir in order to prescribe the correct antibiotic that will kill that species. For the purpose of bacteria identification, numerous tests have been devised to find out the exact species in question. However, because new strains continue to emerge, it is of the utmost importance that microbiologists and microbiology students understand the nature of each bacterial species and how that species creates and maintains its complex communities. Of equal importance is the quick identification of the contaminating source. The purpose of this study is to identify an unknown bacterium using as few tests as possible, and discover its importance in the world at large. Materials and Methods

To begin this study, a slant of bacteria was given by the instructor labeled “unknown 1.” From this culture, three T-streak plates were aseptically inoculated and one was placed in a 37 degrees Celsius incubator, one was placed in a 25 degrees Celsius incubator (to check for optimal growing temperature, as well as purity of the culture), and the other was placed in a gas pak pouch. A reserve culture was also made which replaced the working slant after 21 days of use. After the cultures had incubated for 2 days and tested pure, a Gram stain was performed from the working slant, and an oxidase test was performed using a TSA plate. The gas pak system was used by creating an inoculated TSA plate (using aseptic techniques) and placing it in a GasPak pouch. A generating packet was placed inside the GasPak pouch and the pellet attached to the pouch was observed for a white color, indicating that oxygen was absent. The GasPak was then placed in the 25 dgerees Celsius incubator for 48 hours and observed for growth. The Gram stain was accomplished by preparing a smear and applying crystal violet to it, and allowing it react for 2 minutes. The crystal violet was then washed off by squirting water onto one end of the slide, tilting it and allowing the excess violet color to wash off. The slide was then flooded with Gram's iodine and allowed to react for 1 minute, 30 seconds. The slide was then rinsed again with water using the same technique as before, however this time the excess water was shaken off. The next step was tilting the slide at an angle (hot dog style) and adding acetone-alcohol one drop at a time until the the color came off. It was then crucial to completely rinse the slide with water. After the slide was rinsed, it was time to add the safranin to the slide, to which it was allowed to react for 60 seconds. The excess safranin was then tilted off the slide and washed with water. The slide was then allowed to dry. The oxidase test was performed to find out whether the unknown bacteria had cytochrome oxidase in its electron transport chain. This was achieved using the TSA plate from the 37 degrees Celsius incubator and streaking the bacteria onto an oxidase card using a toothpick. The card was then observed for a blue color within a 20 second time frame. The next test performed was fermentation of lactose. In order to test for lactose fermentation, a tube of phenol red lactose broth was inoculated from the working slant and then incubated at 37 degrees Celsius for 48 hours. After incubation, the phenol red lactose broth was examined for color change. The fifth test performed was to find out if the unknown bacteria could utilize citrate as its sole source of carbon. This was done using an inoculating needle and aseptically transferring the bacteria into a slant of Simmon's citrate agar by stabbing the needle into the butt of the agar, then streaking it across the top of the agar as the needle was pulled out. The tube was then placed in the 37 degrees...
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