July 5, 2011
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 prove the identity of the bacteria. The unknown bacteria was #9. After performing the tests it is determined that the unknown bacteria is Escherichia coli. 3. Methods: All tests were performed using the aseptic technique to assure the cultures were pure and not contamination was present. The inoculation loops and straight wire stab needles were placed over the flame of a Bunsen burner and heated until red hot (sterile) then cooled before placing them into the bacteria to prevent the killing of the bacteria. The open end of each tube of bacteria was flamed once it was open (before inoculation) and before it was closed (after inoculation). (Morello, 9th Edition. 2008)
EMB (eosin methylene blue) agar was used to determine if the bacteria was a lactose fermenter or a non- lactose fermenter. EMB agar is a selective and differential media. The dyes eosin Y and methylene blue found in the medium inhibit the growth of gram-positive bacteria but not the growth of gram-negatives. Lactose fermenters metabolize the lactose in the media and produce acid byproducts, causing a color change in the colony which is dark purple, almost black. Strong acid productions by organisms result in a metallic green sheen. Weaker fermentation of lactose results in colonies with a pinkish-purple color. Colonies that are non-lactose fermenters remain colorless, or at least no darker than the color of the media.
The unknown bacteria #9 was streak diluted on to an EMB agar plate using a sterile inoculating loop. After incubation, the unknown bacteria #9 produced dark purple/black colonies that had a green metallic sheen. This is a typical test result from the bacteria Escherichia coli.
Phenol Red Sucrose broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate (sucrose). Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube. If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed.
The unknown bacteria #9 was added to the phenol red sucrose broth using a sterile inoculation loop. Only a loop full was added and mixed into the broth. After incubating, the unknown bacteria #9 caused the broth to change color from red to yellow indicating a positive result for sucrose. There were bubbles inside the Durham tube which is a positive result for gas production.
H2S (only from SIM) SIM medium is a combination differential medium that tests three different parameters, which are represented by the three letters in the name: Sulfide-Indole-Motility medium. It is a semi-solid agar that is inoculated with a bacterium to test for hydrogen Sulfide, Indole,...