The first step to the unknown is selecting an actual organism. The best way to select a culture is based on a high-quality distribution. Equally important, shaking up the broth tube facilitates in the distribution. Upon selection, a gram check for purity is performed. Step by step instructions for this procedure can be found in Benson’s, Microbiological Applications p. 99. Furthermore, an aseptic technique must be performed for this test and the entire tests following the unknown. The purpose of this test is to differentiate between gram positive and gram-negative bacteria. The key indicator of gram-positive bacteria is a purple stain and a pink stain for gram-negative bacteria. A slide is viewed with a microscope under oil immersion. Equally …show more content…
Use the same plate from the TSA streak. Determine if catalase is produced through the break down of hydrogen peroxide. Proceed by adding reagent hydrogen peroxide and pay attention to bubbling. In other words, bubbling takes place due to breaking down hydrogen peroxide and the fabrication of oxygen gas (Benson, 2010). The control for this test is S. aureus. Blood Agar Plate A streak for isolation is made on the plate. In addition, check plate for hemolysis. Look for the organism’s ability to produce hemolysin. Bacteria with hemolysin will generate different patterns. To start with, partially green around the colony is alpha. Secondly, complete clearing around the colony is betta. Thirdly, cloudiness or no result is gamma. The controls are K. pneumoniae, B. cereus, and S. saprophyticus in order based on previous results. Simmons Citrate Agar Slant The use of this check is to uncover whether or not an organism is capable of exercising citrate as a carbon source. Position a needle of your organism along the surface of the slant. Next, start from the bottom and work your way up with the needle. The indicator that is already in the media is Bromothymol blue. An increase in PH will generate a bright blue for a positive result. The control is E.
One bacterium was gram negative. It underwent four different tests. These tests were the EMB test (Eosin Mehylene Blue), the Sulfur Indole Motility (SIM) test, the Urease test, and the Simmon’s Citrate Utilization test. The EMB test checks for a bacteria’s ability to ferment lactose. This test is accomplished by placing the bacteria on Eosin Methylene Blue agar. The agar is selective for gram negative bacteria and those bacteria that can ferment lactose will have colored growth, usually a metallic green sheen.
In this lab project, the microbiology students were given 2 unknown bacteria in a mixed broth each broth being numbered. The goal of this project is to determine the species of bacteria in the broth. They had to separate and isolate the bacteria from the mixed broth and ran numerous tests to identify the unknown bacteria. The significance of identifying an unknown bacteria is in a clinical setting. Determining the exact bacteria in order to prescribe the right treatment for the patient. This project is significant for a microbiology students because it gives necessary skills to them for future careers relating to clinical and research work.
I began my test to classify my unknown bacteria by performing the Gram staining because according to the first period procedure of the laboratory manual and the Appending H, it was the first test that should be done to plan and proceed to the next tests. Washed bottle of distilled water, three slides, and Gram-staining reagents
Phenotypic methods of classifying microorganisms describe the diversity of bacterial species by naming and grouping organisms based on similarities. The differences between Bacteria, Archaea and Eukaryotes are basic. Bacteria can function and reproduce as single cells but often combine into multicellular colonies. Bacteria are also surrounded by a cell wall. Archaea differ from bacteria in their genetics and biochemistry. Their cell membranes are made with different material than bacteria. Just like bacteria, archaea are also single cell and are surrounded by a cell wall. Eukaryotes, unlike bacteria and archaea, contain a nucleus. And like bacteria and archaea, eukaryotes have a cell wall. The Gram stain is a system used to characterize bacteria based on the structural characteristics of their cell walls. A Gram-positive cell will stain purple if cell walls are thick and a Gram-negative cell wall appears pink. Most bacteria can be classified as belonging to one of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, and Gram-negative bacilli) (Phenotypic analysis. (n.d.).
To record the results of Oxygen created from decomposing Hydrogen peroxide with catalase that are either heated, cooled of left in room temperature.
The purpose of this assessment was to research, design and conduct an experimental investigation on the effect of substrate concentration (manipulated by increasing concentration of pH buffer) of catalysed reactions by measuring the volume of oxygen produced as the reaction proceeded.
The bacteria ferment lactose, then cause the pH of the media agar to drop and resultant change in pH is detected by neutral red. The neutral red is absorbed by the lactose fermenting bacteria and appear as bright pink to red on the MacConkey agar. The strong lactose fermenting bacteria produce resulting in a pink halo in medium while the weaker growing on MacConkey agar will still appear pink to red but will not be surrounded by a pink halo in the surrounding medium. The gram-negative bacteria that grow but do not ferment lactose appear as colourless on MacConkey agar plate. The agar surrounding the bacteria remains transparent. Lactose can be replaced in the medium by other sugars and the abilities of gram-negative bacteria to ferment these replacement sugars is detectable in the same way as lactose
One of the first tests performed was the Gram Stain. Gram Stain differentiates bacterial organisms according to their cell wall structure. Gram-positive cells will stain blue to purple whereas Gram-negative cells will stain red to pink (Lab Handout; Gram Stain). Upon performing the Gram Stain on my unknown cell, I concluded that it was a Gram-positive cell due to the purple color when viewed under the microscope. I also determined the shape to be cocci, as evidenced by the spherical shape of the bacterium.
The slide was placed on a staining tray where the sample was stained using crystal violet. After a minute the sample was rinsed. After that Gram iodine was put on the sample and was rinsed again, after a minute. Following that a 95% alcohol/acetone was dropped on the sample until only a faint violet like color is seen. Immediately following that the slide was rinsed in order to prevent further destaining. Then the sample was covered with safranin for 45 seconds in order to restain the destained gram-negative bacteria making it have a purple-pinkish color. Following this the slide was rinsed and the sample was observed.
The last test performed to assist in determining the identity of the unknown bacteria was a citrate utilization test. This test was chosen to determine if the unknown organism could use citrate as its sole carbon and energy source. It was completed by inoculating the slant of Simmons citrate agar and allowing access to oxygen. A positive result would include a color change in the agar from green to blue, due to the utilization of citrate causing an increase in pH. A negative result would not include a color change in the agar, it would remain
Once the plates incubated for a week long, we obtained the plates and observed what we saw on the plates. This would be known as the macroscopic observations of your microorganisms. We needed to take note on how many colonies were formed, as well as the physical characteristics that appeared on the plate itself. After we noted the macroscopic characteristics it was time to transfer a colony to slides. At this point each member of the group had picked one colony from their own agar plate to transfer to slides via different staining techniques. We completed two separate techniques, the simple staining technique, which is a positive staining technique, as well as the negative staining technique. If there is a possibility that the microorganisms could possibly be a mold, you will need to do a third type of technique called, Lactophenol Blue
Each student in Microbiology, went through a series of experiments to determine the unknown microbe that was given. The microorganism was inoculated to different test that in 48 hours the solution would either change or remain unchanged. Though some of the experiments required additional steps that was done on the next day of lab. Not only was a series of test done, but also a gram stain was recommended during the discovery of the unknown. The gram stain played a major step, if this step was done correctly students could cross out organisms that did not come near to theirs. By looking through the microscope, the morphology of the bacteria was distinguished by the steps of staining the slide correctly and the oil immersion that clearly shows the shape of the bacteria. The gram stain helped to either categorize your microbe as a gram negative/positive rod or gram positive cocci.
The purpose of the microbiological experiment was to applying different types of techniques and methods learned in microbiology lab to a bacteria so that it may be accurately and precisely identified. “The words accurately and precisely are not the same in microbiology, accurately has the meaning of if repeated the same answer continues to be the result, precisely has the meaning of the correct answer.” “Microbiological experimentation often involves test that determine the ability of an organism to use or produce some chemical, or to determine the presents or absence of a specific organism in a sample.” The scientific methods that were performed aided in the precise identity of the bacteria.
Biochemical tests are used to identify microbes in the laboratory to aid in pinpointing the different groups of bacteria. The bacteria vary in the cellular morphology and staining properties as well as structural and metabolic properties. Using biochemical testing, it permits a keener study at related organisms. In addition, the use of numerous color changes that occur with the test, allow to for a rapid identification of comparisons and variances of the bacteria that are tested.
The Catalase Test indicated a positive result. This showed that the bacterium contained the enzyme catalase which converts hydrogen peroxide into water and gaseous oxygen. The positive control used for this test was Micrococcus and the negative control for this test was Entercoccus.