Simmon Citrate Agar

The purpose of this lab was to identify two unknown bacteria from a mixed culture. The reason for identification of unknown bacteria was to help students recognize different bacteria through different biochemical tests and characteristics. This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

Research Question: How does the size of the cell affect its efficiency in exchanging substances through several ways, like diffusion?

The first result of importance was the result of the Gram stain. The observations of the unknown bacteria from the slant culture after Gram staining showed that the unknown bacteria were Gram negative bacilli (Image 1). After determining the unknown bacteria was Gram negative, an oxidase test was conducted on a sample from the slant culture. The cotton swap with the sample of bacteria did not change color when the oxidase reagent was applied, thus providing a negative result. With a negative oxidase test, further tests were conducted to determine various characteristics of the unknown bacteria. A MR-VP broth was inoculated with a sample from a slant culture of unknown bacteria. After incubation, the methyl red reagent was added to the broth, and the broth turned red, providing a positive result (Image 2). An EMB agar streak plate was inoculated with a sample from a slant culture of the unknown bacteria, and after incubation, growth was found on the plate, providing a positive result (Image 3). A Citrate agar slant was inoculated, and after incubation, growth was found on the media, providing a positive result (Image 4). A Urea agar slant was inoculated, and after incubation, the agar had changed from a peach color to a bright pink color, providing a positive result (Image 5). Using the flowchart (Figure 1) developed from the Table of Expected Results, the lab partners started at the oxidase test. Given the negative result of the oxidase test, the flowchart is

In a laboratory setting, it often becomes necessary to identify an unknown organism. In this experiment, researchers classified an unidentified bacterium based on its physical structure, colony morphology, optimal conditions and metabolic properties. A Gram stain using crystal violet, iodine, and safranin and a simple stain using methylene blue characterized the organism’s cell wall. Cultural behavior was classified by inoculating the organism onto nutrient agar and incubating it at 37° C for 48 hours, and observing its behavior, as well as using SIM medium to test for motility. Optimal growth temperature was

The trend I saw in the different concentrations of nutrients was the rod shaped bacteria which be-come more obvious when more nutrients are added. At 0.1g, the agar plate looks mostly smooth but at the 0.6g individual rod, shapes are prominent. Once it gets to 1.3g the colonisation is more crowded, making it harder to see. This seems only visible effect of altering the nutrients is the rod shaped forming and the visibility of them.

Figure 2 is a representation of the average saturation of each cuvette at a specific point time as a function. The y-axis shows the specific saturation points from figure 1, and the x-axis provides the different levels of pH. The pH scale provided on the x-axis ranges from 0 to 14, 0 being the most acidic and 14 being the most basic. The point chosen from figure 1 was the saturation levels of each cuvette at 110 seconds. The saturation point was chosen because in the previous graph at time 110 seconds the reactions of

For the temperature test each bacteria was placed on a nutrient agar and incubated for either 10, 20, 30, 40, or 50 degrees Celsius for 48 hours. During the pH test, each organism was placed on four agars varying in pH level from pH 2, 4, 6 and 8 and incubated near 37 degrees Celsius for 48 hours. For the osmotic pressure test, each organism was placed on four agars one each containing 2%, 5%, 8%, and 11% NaCl concentration levels. These were incubated near 37 degrees Celsius for 48 hours. The results of the tests are recorded in Tables 1, 2, and 3. All tests were performed according to the instructions provided in Leboffe & Pierce(1). The biochemical tests used on both unknowns and the ubiquity are:

The optimal temperature of Bacillus lichenformis bacterial amylase and Aspergillus oryzae fungal is determined by mixing a starch solution into the bacterial and fungal amylases that are put in four different temperatures (0, 20, 55, 85 degrees Celsius). Then after every two-minutes, ending at the ten-minute mark, a small sample of the starch-amylase mixture is put into a well with a couple drops of iodine to help show the change in starch. This was done because when iodine is exposed to starch it changes color. Based on the color chart given in our lab manuals, the reaction of the amylase to the starch solution will give the starch-amylase mixture in the iodine a yellow color to signify if the presence of solely iodine and/or little starch depending on temperature. This means that the amylase broke down the starch solution because its temperature was optimal. Majority of the results came out black or dark brown therefore the amylase wasn’t put in the proper temperature to break down the starch solution at a faster pace. The temperature that seemed most optimal was at 55 degrees Celsius for both fungal and bacterial because it showed a more brown to yellowish color when put into the iodine. That showed that the amylase was able to break down the starch at a faster rate because it was working at its optimal temperature.

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

Bacteria groups or species can be differentiated by the fermentation patterns. The end-product of carbohydrate fermentation is an acid or acid with gas production and is dependent on the organism involved in the fermentation process. The carbohydrate fermentation tests detect if an organism is able to utilize glucose, lactose and sucrose. Phenol red is used as a pH indicator because it can indicate a change in pH when acid products are formed. Bacteria can utilize certain sugars resulting in an alkaline by-product which changes the color of the carbohydrate broth from red to yellow. Bubbles trapped within the Durham tube indicate the production of gas. The Phenol red carbohydrate fermentation tests determine that my organism E. coli can utilize glucose, lactose and sometimes sucrose but can only produce gas in glucose and lactose. (Phenol red carbohydrate fermentation lab

First, three titration curves and three second derivative curves were created to determine the average pH at the half-equivalence point from the acetic acid titrations. Titration curves were used as visuals to portray buffer capacity. The graphs and a table, Table 1, that showcased the values collected were created and included below. The flat region, the middle part, of Figures 1, 2 and 3, showed the zone at which the addition of a base or acid did not cause changes in pH. Once surpassed, the pH increased rapidly when a small amount of base, NaOH, was added to the buffer solution. Using the figures below and

2. Introduction: Each student was given unknown bacteria and was instructed to perform a variety of experimental tests that would help to identify their bacteria. During the process of identification, the unknown bacteria was added to many different testing medias using aseptic technique. They are as follows: lactose fermentation on eosin methylene blue (EMB), TSI (Triple Sugar Iron agar), Phenol red sucrose, the SIM test, H2S by SIM, IMViC (indole, motility, voges-proskauer, and citrate), Urease (urea broth), PDase (Phenylalanine Deaminase), Lysine Decarboxylase, and Ornithine Decarboxylase. Colonial morphology on EMB was used to

Each mixed culture that was tested had one gram positive and one gram negative bacterial species. The possible species of bacteria that could have been isolated from the mixtures included the following: Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Enterobacter aerogenes, Salmonella enterica, and Pseudomonas aeruginosa. The identities of the unknown species were determined through comparing the experimental data against data acquired from earlier experimentation.

Starch is a polysaccharide made up of glucose molecules. Some bacteria have an enzyme called amylase which breaks starch down into glucose subunits. The Starch Hydrolysis test is used to determine the production of amylase. Iodine, which is the mordant used in Gram staining, is used in this test to detect the presence of starch. In order to do the Starch Hydrolysis Test I first inoculated a starch plate by using aseptic technique. I streaked the middle of the starch plate with the bacteria into a single line. Then, I let it incubate overnight at 37°C. In week 2, after I added several drops of Gram’s iodine to the starch plate it was ready to be analyzed for starch hydrolysis. The iodine complexes with starch to form a blue-black color in the culture. Clear halos surrounding colonies is the result of their ability to digest the starch due to the presence of amylase.

The purpose of the two experiments was to determine the fundamental effects that temperature has on the growth and survival of bacteria. During the first experiment five different bacterial broth cultures of Escherichia coli, Pseudomonas fluorescens, Enterococcus faecalis, Bacillus subtilis and Bacillus stearothermophilus were individually incubated at temperatures of 5, 25, 37, 45 and 55°C for one week in an aim to distinguish the effect temperature has on growth and survival of the five different species. After one week they were observed for distinguishable changes by the turbidity showing an indication of bacterial growth, or the clarity an indication of no survival.