Glucose / Meltrose, Lactose, And Sucrose Experiment?

An unknown bacterium was handed out by Dr. Honer. The appropriate tests were prepared and applied. The first procedure that was done was the gram stain. Under a microscope, if the gram stain is purple, the bacterium is gram positive, if the stain is red, it is gram negative. The next test was the fermentation tests for glucose, sucrose and

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.

The purpose of this lab was to test different substances using various procedures to see what biomolecules were present and ultimately find out what restaurant Anna Lyza had eaten at before she died. For the first control test, we used vegetable oil to test for lipids. So, if the solution does not contain lipids, it does not become translucent when placed onto a paper bag square and held up to a light. So, it is a negative result. However, in the presence of lipids, the solution will become translucent when placed onto a paper bag square and held up to a light. Therefore in this case, the result is positive. On the other hand, we used albumin egg to test for proteins in another control test. If the solution does not contain proteins, it will not experience any color change and so it is a negative result. When there are proteins existing in the solution, it will turn bluish/purplish and for this reason it is a positive result. Furthermore in the third control test, we used dextrose to test for simple carbohydrates such as glucose. If the solution does not contain simple carbohydrates, it will not undergo any color change and will remain a blue color. So, it is a negative result in this circumstance. If there are simple carbohydrates present in the solution, the solution will turn reddish and so the result is positive. For the last control test, we used starch solution to test

This test is used to detect if the bacteria contains any deoxyribonuclease activity. Because no color change was observed from blue to clear my unknown bacteria displayed a negative result.

To perform this test, a tube of broth rich with glucose is acquired. In this tube is phenol red, a pH indicator. Initially, the tube appeared pink in color, indicating a normal pH level. Next, a sample of unknown #44 is introduced into this medium using the aseptic technique, and this is allowed to sit for several days. If the organism is able to ferment glucose, the pH in the medium would decrease and cause the phenol red to exhibit a yellow color. In addition to the straw color, gas can also be produced and trapped inside the Durham tube placed in the medium. This production of acid and gas is a direct result of the fermentation of glucose, as seen with unknown

The filter paper was then observed to see if it changed blue or not, in order to see if the bacteria produced cytochrome c oxidase. The final test used in the experiment was an API test. To begin the API test, a solution with bacteria and 5 mL of sterile saline, had to be made with a turbidity the same as the McFarland No. 3 (BaSO4) standard. This was done by adding loopfuls of bacteria to the saline solution, mixing the solution on the vortex, and then comparing the turbidity to the McFarland No. 3 standard, until the tubes were both at the same cloudiness. This created solution was then used in the API test by adding specified amounts to each of the microtubes on the API strip. For each of the microtubes whose names were not underlined or boxed, the tubes were filled to where the microtubes met the capsule. In the microtubes whose names were underlined, the microtubes were slightly underfilled, and then the capsule was filled with mineral oil in order to create and anaerobic environment. The last of the microbes were the ones whose names were boxed. In each of these the microtube and the capsule were filled all the way up with the bacteria. The API test strip was then placed in the 37°C incubator for 20 hours. After this time, observations were made about each of the different microtubes based on a given summary of results chart for the API test. A select number of microtubes had

The next two tests that I performed in the laboratory were the oxidase and catalase tests. The oxidase test is used to determine if a bacterium produces certain cytochrome c oxidases which can transfer electrons to oxygen. This experiment requires an oxidase test reagent that when introduced to the bacteria will turn purple indicating a positive result (Lab Handout; Oxidase Test). The result of this experiment was no color change

This test should be used when trying to identify organisms that produce catalase. It is used when differentiating between Catalase positive micrococcaceae and catalase negative streptococcaceae and some variations of the catalase test are for mycobacterium.

The unknown was also tested for catalase. Aerobic bacteria produce the enzyme catalase. It breaks downs hydrogen peroxide into water and oxygen. After smearing an isolated colony of the unknown on a slide, hydrogen peroxide was added and it resulted in bubbling. Bubbling suggests a positive test and if there is no bubbling, it is a negative test. A citrate lyase test was also done using citrate agar. Bacteria

To do this, one must use a series of biochemical tests. These tests will investigate the bacterium’s ability to use available enzymes and degradation of specific carbohydrates, lipids, proteins and amino acids. The use of these organic compounds will produce byproducts that can be observed to determine the metabolism of the unknown bacterium. One of the first biochemical tests that I prepared was the S.I.M. test, which is a combination of three tests in one test tube. The “S” represents sulfide for the sulfur reduction test. This individual experiment detects hydrogen sulfide production. If the unknown bacterium has either cysteine desulfurase or thiosulfate reductase it will be able to reduce sulfur to a hydrogen sulfide gas. Once H2S is produced, it combines with the ferrous ammonium sulfate, forming a black ferrous sulfide precipitate that can be seen along the original stab line; the presence of black along the stab line would indicate a positive result. This test can also be used to detect the presence or absence of motility. This observation is what makes up the “M” in the S.I.M. test for motility. The only thing that needs to be done for this test is observation. If there is growth away from the stab line, then that indicated that the unknown bacterium is motile. If there is no growth away from the stab line, then that indicates no motility and a negative result. Unk#19 presented with black precipitate as well as growth away from the stab line. After completing the motility and sulfide tests, I could then perform the indole test. The purpose of this test is to find if the unknown bacterium contains the enzyme tryptophanase can break down the amino acid, tryptophan, into its three metabolic ingredients: indole, pyruvic acid, and ammonia. If the bacterium contains tryptophanase, they will then use ammonia and pyruvic acid for nutritional needs,

Once observing the bacteria with the microscope at the highest magnification of 1000 oil immersion each student should know if the unknown bacteria is gram positive or negative as well as if the microorganism morphology is coccus which is cells sphere shape or bacillus rod shape. If the microorganism is gram positive under the oil immersion should show purple, but if gram negative pink should show up. Proceeding to the catalase test by taking a slide and using a sterile loop put a small sample of bacteria on a clear glass slide then place a couple drops of the hydrogen peroxide on the bacteria. Afterwards students are able to choose which media would be best to get results to identify the unknown bacteria.

The next experiment from the flowchart is the Acid and Gas from Lactose, but in our lab manual it is call Phenol Red Fermentation Broth test. This experiment allows for us to see if the microbe ferments a sugar with a distinctive color change. A special test tube called Durham tube is used to detect if they are also gas producers.

In the Sugar Fermentation experiment I ended up with three negatives, being Mannitol, Sorbitol, and Arabinose, and four positives, Lactose, Glucose, Sucrose, and Maltose. In the Litmus milk project, my bacteria turned out to be top neutral with reduction in the bottom. I had no coagulation, no peptonization, negative for Acid, and negative for Alkaline. A few other tests indicated I was positive for Arginine and Lysine, but I had tested negative for Bile Esculin. The bacterium tested was positive for all of the salts as well as for the TSI agar and the

For the MR Test, 3 drops of Methyl Red Reagent was added to one of the test tubes and the color was observed. The color changed from yellow to red to confirm a MR positive result meaning this microbe could ferment glucose into mixed acids and lower the pH. For the VP Test, 15 drops of VP-Reagent “A” was added and mixed then 5 drops of VP-Reagent “B” was added and color was observed after 10-20 minutes, undisturbing the test tube. The analysis revealed no red top layer, a negative result, which suggests this microbe cannot ferment glucose into acetoin. For the Indole Test, 5 drops of Kovac’s Reagent was added to the green-capped test tube (Figure 5) and the top layer was to be observed. No red layer appeared, another negative result, which confirms that this microbe does not produce indole from tryptophan. The Citrate test tube appears to be positive from Figure 5 since it is blue with growth, however after confirming my results with the instructors, it is a false positive. Thus, this microbe cannot utilize citrate as a sole carbon source and the false positive could have occurred from contamination or another unknown

Discussion The purpose of this experiment was to produce ethanol through fermentation of sucrose. Once the solution had fermented for a week, it was subjected to fractional distillation to remove the ethanol from the supernatant. As seen in Table 1, while the first fraction contained a moderate concentration of ethanol, the second and third fractions did not contain any ethanol at all. This can be attributed to the fact that there was little to no ethanol in the supernatant obtain from the fermentation solution.