Exercise 4 : Use Selective And Differential Media

A mixed culture of two unknown bacteria was provided by the instructor. The methods used for

In the beginning of lab, we were advised to obtain a nutrient agar petri plate, which is used for the cultivation of microbes supporting growth of non-fastidious organisms. Since it contains many nutrients, a wide variety of bacteria and fungi can grow. Taking the plate,

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

(Biology Dept.). 0.1 ml of E.coli K or 0.1ml of E.coli B was added to the 10 fold dilution. Using soft agar technique, the growth media mixture with E.coli was plated and incubated.

The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible

The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. The differential tests used to identify the unknown cultures were oxidase, catalase, lactose and sucrose fermentation, Kugler/iron agar, nitrate reduction, gelatin hydrolysis, starch hydrolysis, manitol salt, MR-VP, citrate, bile esculin,

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

An unknown bacterium 15 was awarded and labeled at the table ready to be identified. Using the skills and test that are taught and learned in microbiology were applied into learning what the unknown bacteria culture was. There were multiple procedures and test done in order to gain all the information needed to determine which bacteria was given. In order to find what the bacteria was the first step was finding the right environment and temperature that would allow the bacteria to thrive and grow. Determining this is one of the most important steps in being able to obtain conclusive results that would allow the results of the test to be accurate and correct. Without the correct temperature and environment the bacteria will give inconclusive results which will alter and skew the end results and may lead to the wrong conclusion. By using the methods that were obtained and learned through the microbiology class allowed the skills and knowledge to determine the bacteria and execute the tests in order to determine the culture.

When reflecting back to experiment 3, Aseptic Technique and Culturing Microbes, I realized the large amount of microorganisms that can be found in everyday life. Many different types are found with in the human body. Theses experiments focused on two types of bacteria. First was Staphylococcus epidermidis, found on the skin, and second was Lactobacillus acidophilus, found in the gastrointestinal tract. Both have similar needs for growth when it comes to temperature, however, different growth environments are used.

Being able to control bacterial growth is something that is important in our everyday lives. As shown in the previous labs, bacteria can grow and create colonies extremely quickly especially in the right environments. By acknowledging this, it is then important to get an understanding of how bacterial growth can be controlled by humans. To control microorganisms it means to inhibit their growth (static) and or kill them (cidal) (Kenneth Todar, 2015); therefore since focusing on bacteria the terms bactericidal and bacteriostatic are both extremely important for this lab. One broad method we will use to control bacterial growth is heat. The amount of heat needed to control bacterial growth is different for different species of bacteria (Kenneth Todar, 2015). Bacteria can also respond differently depending if moist heating method such as an autoclave with steam is used, or a dry heating method such as inoculating a loop over a fire is used (Kenneth Todar, 2015). UV works by damaging the cells DNA, without proper DNA, the cells will die and the object

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.

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.

This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.

Broth cultures of E.coli, Micrococcus luteus and Vibrio natriegens were streaked onto the respective sections. The plate was then incubated at 37oC for 48 hours. The process was then repeated on nutrient agar plates with 5, 6.5 and 10% salt.