The purpose of this experiment was to try and identify an unknown microorganism by performing a series of biochemical tests on a pure bacterial culture.
Materials and Methods
Carbohydrate fermentation: For the carbohydrate fermentation lab there were two fermentation tubes, each with nutrient broth, a single carbohydrate, Phenol red (PR) which is a pH indicator that is red in basic conditions and turns yellow in acidic conditions, and an inverted Dunham tube. There was one tube with sucrose as the carbohydrate and another with lactose as the carbohydrate. The tubes were inoculated with a pure culture of unknown #3 using aseptic techniques by obtaining a loop of the culture and adding it to the liquid medium in the tubes. The tubes were then incubated in a 37° C incubator until the second lab. The color change of the pH indicator was used to determine a positive or negative result. If the medium turned from red to yellow, then acid was produced. If the medium turned yellow and there are bubbles in the Dunham tube, then there was acid produced as well as a gas. Whereas if the medium remained red then no acid was produced and if the medium turned a deep magenta, then ammonia was produced from the utilization of protein.
Test for amylase through starch hydrolysis: In order to test for amylase activity, an inoculation of unknown #3 was added in a straight line to the surface of a starch agar plate using aseptic techniques using a loop. A starch agar plate is made up of nutrient agar and starch. After inoculating with the organism, the plate was incubated in a 37° C incubator until the second lab. The plate was then flooded with iodine. Iodine reacts with the intact starch on the plate and forms a blue complex. After flooding, it is a positive result if the starch was hydrolyzed causing the iodine to not form a blue color complex leaving the plate clear around the bacterial growth. Whereas the starch-iodine complex will form if the result is negative, causing the intact starch to turn from clear to a dark blue color meaning that the starch has not been hydrolyzed.
Test for tryptophanase through indole production: In testing for indole, an aseptic inoculation of unknown #3 using a loop was added to a medium of 1% tryptone broth. This tube was then incubated in a 37° C incubator until the second lab. In order to determine if indole was produced from the breakdown of the tryptone, during the second lab, 5 drops of Kovac's reagent were added and the tube was gently shaken before letting it stand for 10 minutes. The presence of indole causes a positive result. If a red ring forms at the top of the tube indole is present. Whereas if the results were negative, the reagent at the top of the tube would remain yellow.
Test for motility and sulfide production: A culture tube containing sulfite indole motility (SIM) medium was inoculated with a pure culture of unknown #3 using aseptic techniques by stabbing the SIM with an inoculation needle that has unknown #3 on it. The tube was then incubated at 37° C until the second lab. If H2S is formed it reacts with ferrous salts to form black salts. Upon examining the medium for growth, if there is a black precipitate in the medium then unknown #3 is positive for sulfide production. Also when examining the medium if you can clearly see the line in which you stabbed the SIM medium, then the organism is not motile whereas if there is movement around the exact location of where you inoculated the SIM, then the organism is motile.
Test for urease through urea hydrolysis: A tube consisting of yeast extract, urea, and phenol red a pH indicator. Urease splits urea into ammonia and carbon dioxide. Using aseptic techniques a loop full of pure culture of unknown #3 was added to the urea broth tube and incubated in a 37° C incubator until the second lab. If the hydrolysis of urea in the medium splits into enough ammonia, the medium becomes an alkaline...
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