Unknown Lab Report

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.

The purpose of this lab was to identify an unknown bacteria culture using differential tests. The identification of the unknown culture was accomplished by identifying the bacteria based on its specific metabolic characteristics and morphology. It is suggested that culture 11 is a sample of Enterobacter aerogenes.

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.

Nitrate reduction was tested for by inoculating a nitrate broth with the unknown gram (-) culture, and allowing growth to take place. Adding 2 drops of both sulfanilic acid and α-napththylamine to the medium if the first test to see if nitrite is present. If nitrite is present, the medium turns red, indicating a positive test. However, if the medium does not change, a second test is performed to see if nitrite was further reduced. In this second test, zinc powder is added to the broth to catalyze the reduction of any nitrate present to nitrite. If nitrate is present when the zinc is added the reduction of this compound will cause the medium to turn red, from the previously added reagents. Red medium on the second addition indicates nitrate was not reduced and a negative test result. However, if the medium does not change after the addition of the zinc, the unknown is positive for nitrate reduction, as the nitrite has just been further reduced, preventing its detection. The result that yielded was positive on the first step.

The enzyme urease breaks urea down into NH3 and CO2. An orange broth containing urea is used for this test and needs to be inoculated with the gram negative bacteria. A pink color in the medium indicates a urease-positive organism, an orange or yellow is negative.

I used an inoculating needle to stab the SIM test tube and then incubated it at 37 degrees Celsius for 24 hours. The SIM test was used to test whether an organism has the ability to reduce sulfur to hydrogen sulfide. Iron salts in the media reacts with the hydrogen sulfide to form a black precipitate called ferric sulfide. If sulfur can be reduced than a black color will be seen in the tube. This test also sees if an organism is and indole producer. Indole producers are bacteria that produce the enzyme trytophanase which can hydrolyze tryptophan to pyruvate, ammonia and indole. To test for indole production,

A gelatinase test was performed using nutrient gelatin to determine the ability of the bacteria to produce gelatinases. The gelatin was inoculated with the unknown cultures and incubated. The tubes were then placed on ice to reform the warm gelatin. If the gelatin remained solid after inoculation, the bacteria did not produce the gelatinase enzymes to liquefy the agar, constituting a negative test. However, if the gelatin was liquefied, the enzymes were secreted and the bacteria were positive.

The oxidation fermentation test was used to differentiate if the organism utilizes lactose, mannitol, glucose and citrate aerobically (oxidation) or anaerobically (fermentation). A methyl red test was performed to determine if the organism carried out mixed-acid fermentation when supplied glucose. A Voges-Proskauer test was performed to evaluate if the unknown was able to ferment glucose into butanediol. A citrate test was performed to determine if the unknown organism was able to break down citrate into ammonia. An oxidase test was then performed to determine if the unknown culture was oxidase positive or negate.

Table 4 is showing test results for Phenal Red fermentation broths, Methyl Red-Voges Proskauer broths, Catalase, oxidase and Urease. In the three Phenel Red tubes the unknown bacteria #3 tested positive yellow for fermentation. However, when reagents were added in the MR-VP tubes the Methyl Red turned a Pinkish color detecting the unknown bacteria #3 strong acid formed and VP tube remained yellow detecting no formation of acetoin. The positive catalase test was indicated by bubbles when peroxide was mixed with the bacteria. However, the negative oxidase test indicated no cytochrome oxidase is present. The final test in the table is the Urease test. The result of the Urease was positive pink color detecting the production of

Different strains of E.coli can be isolated and identified by a variety of biochemical tests. The biochemical tests commonly used to identify E.coli include: lactose, oxidase, indole, and beta-glucuronidase tests. The use of lactose to identify a strain of E.coli is an appropriate method since intestinal bacteria, such as E.coli, typically undergo lactose fermentation. Using MacConkey agar, microbiologist are able to analyze if lactose is fermented and detected by the change of color in the medium. The oxidase test is used to identify if the bacteria synthesizes the enzyme cytochrome c oxidase that is part of the electron transport chain. When the enzyme cytochrome c oxidase is present, the reduced reagent, tetramethyl-p-phenylenediamine will donate its electron to cytochrome c oxidase and a purple color end product will show in the media. The indole test is performed by microbiologist to determine if E.coli is capable of converting tryptophan to indole. Lastly, beta-glucuronidase test is used to determine if an organism produces the enzyme glucuronidase by hydrolysis of nitrophenyl-beta-glucopyranosiduronic acid. In the presence of glucuronidase, the medium develops a yellow color (Trepeta & Edberg,

The first a catalase test uses hydrogen peroxide to see if it can be reduced to oxygen and water by the presence of bubbles. The next test, Bile Esculin, which is an Esculin medium that if the bacteria reacts with the ferric chloride the agar will turn black. The last test being used is a SIM test which is a few tests in one. It tests for sulfur reduction, which would turn black; motility, which shows growth around the incision and indole production, which is the removal of an amino group and its reaction with the Kovacs reagent turning a red color (Allen, 2016). The whole premise of this lab is to take an unknown sample and try and separate it into both Gram-positive and Gram-negative bacteria.

Ammonia builds up in the medium when urea is hydrolyzed. The buildup of ammonia changes the pH of the medium to alkaline. The increase of alkalinity causes the pH indicator, phenol red, to change from an orange-red to a bright pink indicating a positive for urease. I sub cultured my bacteria in a tube containing urea and phenol red. My urease test was negative.

Assays are used as an indicator of whether or not the purification process is working correctly, or if the proteins have been destroyed in the process (1). The lab consisted of purifying wheat germ by utilizing the enzyme that is already present in the substance itself, acid phosphatase. Acid phosphatase was being separated from the wheat germ using a centrifuge (a machine that spins at high speeds in order to separate substances with different densities in a solution) during each step of the experiment to continuously separate and purify the sample (3). There were three assays in which were used during the experiment. One assay used during the experiment was a Bradford dye reagent, which was a blue dye that was added to samples of the supernatant to display the different concentrations of proteins present in each. Another assay that was used during the lab was the KOH. This reagent was added to different samples of the supernatants than what were used for the Bradford dye. The KOH was used to help stop the reaction that had originally taken place from the addition of the acid phosphatase reagent in step three, but its main use was to distinct the different concentrations of the proteins present in each step of the purification

The gram stain, when viewed under a microscope, determined that the bacteria were a gram positive coccus. The bacteria were not endospore formers, as determined by the negative results of the desiccation test, indicating that the bacteria had died. When in contact with hydrogen peroxide, the bacteria rapidly began producing bubbles, giving a positive result for the oxidase test. The results of the SIM test were negative for motility, as seen in the lack of growth throughout the tube. Due to the growth present inside the GasPak, the bacteria were revealed to be facultative anaerobes. The results for each carbohydrate fermentation came back positive, as all tubes had changed from red to yellow, indicating fermentation of each of the carbohydrates. When a colony was spread onto filter paper covered in oxidase reagent, the culture did not turn purple, indicating a negative result for the oxidase test. The ONPG test also yielded negative results, as the salt stayed transparent instead of turning yellow. The culture grown in the water bath showed the bacteria could grow at 45°C, and the cultures grown on the P-agar plate were larger than 5mm. The urease test resulted in a weak positive outcome on the slant, and a negative result in the bottom of the

The simmon’s citrate is use to determine wether the microorganism uses citrate as the carbon source for their metabolism. If organism utilize citrate then positive result is indicated by color change from sea green to blue. However, an unknown microorganism stayed sea green color which is negative for this particular test. In h2s test, cysteine is degrade by cycsteine desulfarase enzyme into h2s and pyruvic acid. The positive result shows the black butt in the bottom of the tube for an unknown organism which indicated the production of hydrogen sulfide. whereas, negative results remains yellow. The urease test played vital role in identification of an unknown microorganism. After performing urease test on an unknown microorganism, Out