Bacillus Badius

The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.

This experiment was centered on metabolic and biochemical testing procedures. The rationale of performing these tests was to distinguish six different microbes from one another and to compare how their metabolic and biochemical processes differ from species to species to determine the unknown sample.

0.0375 mg/ml Porcine Pancreatic Amylase Solution (amylase powder in 0.9% NaCl ), Iodine Solution; each solution were pipetted into each of the 5 test tubes with 5 ml of 1% starch. Each tube contained a 1% starch solution with a different pH. All tubes were at room temperature. Room temperature was 22C. 0.2 ml of porcine pancreatic amylase solution was then pipetted into each tube. A timer was started and every 3minutes the starch / amylase mixture were pipetted from each tube and pipetted into the spot plate for every sample tube, then the iodine solution were added to a spot plate cell for each sample. Iodine reacts with starch to change from yellow to deep blue /black in the presence of starch. A lightening of the blue/ black to a brown color will occur as less starch is present. Results were reported as (+) for presence of starch in the sample or (–) for the absence of starch. After every three minute increment had passed, these same

11. An important tool available in the Virtual Unknown program is the Identification Matrix. From the portion of the identification matrix shown in the Identifying Bacteria tutorial, identify at least one bacterium that has a positive result to the arabinose fermentation test. (1 pt)

I began by running the starch test, which tests for the presence of starch hydrolyzing enzymes. After doing a one-line inoculation of the organism, the plate had to be incubated. Once I received an appropriate amount of growth I added the reagent iodine. The iodine turned the plate purple, formed no clear zone, and lifted the organism off of the plate, which revealed that the starch was not degraded and the enzyme was not present. The organism being lifted off the plate is unique to the bacteria Corynebacterium xerosis indicating that it was my gram positive rod. For reassurance, I ran the Phenol Red Glucose test, which tests if the organism contains various enzymes that determine if the bacteria can ferment glucose. After incubation, the broth turned orange, but this did not provide a clear positive or negative result so I ran the Nitrate Broth Reduction test. The Nitrate Broth Reduction test detects if the organism utilizes nitrate. After incubation for forty-eight hours I added Nitrate A and Nitrate B indicators. However, there was no color change indicating that the test was inconclusive. Since the test was inconclusive, I proceeded to the following step, which included adding a small amount of zinc to the broth, and this turned the broth a red color. The red color indicated that

In the Voges-Proskauer test, I inoculated the tube with bacteria from my TSA plate and incubated the tube at 37 degrees Celsius for 3 days. After three days I placed some Barrits Reagent A and Barrits Reagent B in the test tube. The color change of red or pink indicates a positive reaction for acetoin which tells you that the organism is a butanediol fermentor.

As the flowchart shows, a series of tests were conducted to identify the unknown bacterium #65. Microscopic observation of the gram stain indicated a gram-positive coccus bacterium. S. epidermidis was used as the gram-positive control while E. coli was used as the gram-negative control. This observation led to the elimination of all gram negative and rod-shaped genera: Enterobacter, Citrobacter, Klebsiella, Escherichia, Pseudomonas, Serratia, Alcaligenes, Neisseria, Proteus, Salmonella, Shigella, Erwinia, Veillonella, Flavobacterium, Bacillus, Arthrobacter, Lactobacillus, Listeria and Kurthia (2). By performing the catalase test, it was determined that the bacterium was catalase negative and it did not produce bubbles. M. luteus and E. faecalis were used as positive and negative controls, respectively.

Test tube 1 and 2 didn’t show any color change. These two test tubes had lower levels of pH. Therefore, the enzymes could not change the starch to sugar.

enzymes that will be used during this lab to test the ability of amylase to break down starch ,a

I believe the bacteria in tube 30A is E. faecalis, and the bacteria in tube 29B is E. coli. For tube A, the DNase test yielded a negative result, with absolutely no clearing on the agar. Only three bacteria would produce a negative result: S. epidermidis, E. faecalis, and M. luteus. From there, I performed a catalase test, in hopes of attaining a negative result. The result of the catalase test was, in fact, negative. This eliminated both S. epidermidis and M. luteus because these bacteria would have yielded a positive result. The result of the catalase test was completely negative, as evidence by no bubbles being produced after the addition of a drop of hydrogen peroxide. For tube B, the first test I performed was a EMB plate test. The result

Crystal violet stains the cell of the bacteria and is placed over the heat fixed slide for one minute then rinsed with H2O. The mordant that binds the stain is gram’s iodine and is recommended to be present on the slide for one minute. Alcohol is used as the decolorizer and is dripped over the recently rinsed slide until the slide runs slightly blue. If the bacterium retains the crystal violet it is due to the thicker peptidoglycan in the cell wall (gram-positive). The bacteria will then be stained with the counterstain safranin for 45 seconds. When crystal violet is not retained in the cell wall of the bacterium it will stain pink (gram-negative). A gram stain was immediately performed with the recently inoculated streak plate. An inoculating loop was used to gather some of the bacteria from an isolated colony. The loop was placed on a new slide and the transferred bacterium to the slide. The slide was dried, heat-fixed, and the gram stain process just described was completed. After microscopic analysis, it was determined the first unknown bacteria was a gram-positive cocci. The second unknown was determined to be gram-negative cocci. With this information a set group of test decided and performed based on the Unknown Bacteria Flow Chart as provided in the lab manual (Pg.) these test and the results are shown in record on the following pages. These are separated for the gram positive and the gram negative

In class, we were given the task of identifying an unknown bacterium broth culture. After receiving number 69, I went through several tests to figure out what bacterium I received. First, I created a slide from my broth by putting a small amount of the unknown broth on to a clean slide and letting it dry for ten minutes. After this, I stained the slide by applying four reagents in order; crystal violet, grams iodine, decolorizer and safranin. From the stained slide, I discovered that this bacterium was gram-negative, which would determine the next couple of tests I would do to identify my unknown bacterium. I began by streaking for confluent growth from my broth culture onto a TSA plate. From the TSA plate, I aseptically transferred a loop

This experiment consisted of setting up a control group of starch in various temperature and then placing both fungal amylases and bacterial amylases in a mixture of starch and placing the solution of amylase and starch in various temperatures of water. After a certain amount of time- different amount of time needs to be used in order to have reliable results- iodine is added in a well on spot plates, then two drops of the mixture of amylase-starch is added from each temperature used, by adding iodine into the plates the mixture will show how much starch was hydrolyzed, this is used to calculate the amount of

For this experiment, we have to prepare our phosphorylase which extracted from a potato. We prepared by weighed about 250 grams of peeled potato and cut it into cubes. The extracts then blended with 100mL of 0.1M NaF. After filtered the contents into a clean 250mL centrifuge bottle, we centrifuged it for 3 minutes. Then, separated the supernatant into a centrifuge bottle, which is our phosphorylase preparation. The enzyme assay used in this experiment today is the iodine test. As the iodine reacts with starch, it will form a brown, blue or black precipitate due to the iodine ions forcing into a linear arrangement. The endpoint of the enzyme reaction indicates the presence of starch by using the iodine test to determine. The faster the endpoint is reached, the less active the phosphorylase is.

2cm of a solution was tested and added 2 cm of 10% of potassium hydroxide solution and the test tube was shaked.