The main idea of this experiment was to correctly identify the unknown bacteria, #3. Identification of unknown bacteria yields multiple benefits in many different areas in the research of microorganisms. In this experiment I performed many different test dealing with things such as the presence of enzymes, fermentation abilities and different chemical reactions. Observations made from the tests were then compared to a gram negative unknown chart in order to identify the bacteria. Based off of my results and the chart, I concluded the bacteria #3 was the bacteria Escherichia coli. E. coli is most commonly found in the intestines of warm blooded organisms. Most E. coli strands are non pathogenic however, there are strands known to cause food poisoning.
There are multiple reasons for identifying unknown bacteria. In research, it is a must to be able to identify unknown bacteria if you are comparing the different microorganisms. The identification of unknown bacteria may ultimately lead to the discovery of a new species because the results of the tests performed on it may not match those of any other. In my case I had a gram negative bacteria. Gram-negative bacteria are colored red or pink when Gram stained. They have an outer membrane containing lipopolysaccharide that is not found in gram-positive bacteria. This outer membrane functions as an added layer of protection, by shielding the bacteria from several antibiotics, dyes, and detergents that would damage the inner membrane or cell wall. In this particular experiment, I was issued an unidentified organism that could be one of six different bacteria. In order to identify it I was required to run a series of tests and do a comparative analysis. I received my bacteria in a test tube and I inoculated a TSA plate using the T-Streak method in order to isolate the bacteria and also to grow bacteria to use for my tests. Materials and Methods
The materials I had available to me for use for this experiment were a TSA plate, a TSA slant, a Gelatin Tube, a Methyl Red Tube, a Voges-Proskauer Tube, a Urea Tube, a SIM Tube, a Citrate Slant and a TSIA slant, a light microscope, an inoculating loop, and inoculating needle, a burner, an apron, and some glass slides.
I did a Gram Stain in order to assure that my bacterium was gram-negative. I put a drop of distilled water on a microscope slide and inoculated the bacteria in it. I then heat fixed the bacteria by passing it over a flame three times. Then I applied Crystal Violet to the slide for one minute, and rinsed with distilled water. The slide was rinsed with 95% ethanol for five seconds and immediately rinsed with distilled water. Safranin stain was added to the slide for two minutes followed by a rinsing with distilled water. A light microscope was used at 400x magnification to observe the stained slides.
I used the test tube of my unknown bacteria and an inoculating loop to inoculate my TSA plate and TSA slant. I used the T-Streak method on the plate by flaming the loop and streaking quadrant one than flaming the loop again and streaking quadrant two and then flaming the loop again and streaking quadrant three. I then flame the loop again and inoculated the slant. I than incubated them at 37 degrees Celsius for 24 hours and used them to complete the other tests.
Next I performed the Gelatinase Test using a gelatin tube. I first flamed an inoculating needle and then gathered some colonies from my unknown test tube and stabbed the gelatin tube. This is a differential medium that tests an organisms ability to produce an exoenzyme called gelatinase that will hydrolyze gelatin.
For the Methyl-Red test I inoculated the test tube with bacteria from my TSA plate and incubated the tube at 37 degrees Celsius for 24 hours. After incubation I placed several drops of the pH indicator Methyl-Red into the test tube and observed the color change of either red or yellow. This...
References: * What is E. Coli?. From http://www.medicalnewstoday.com/articles/68511.php
* E. Coli Infection. Fromhttp://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview
* Welcome to Microbugz. From http://www.austincc.edu/microbugz/gelatinase_test.php
* Triple Sugar Iron Agar. From http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/tsia.pdf
* Microbiology Lab Handouts. Lab Manual Dr. Michelle Badon
* Microbiology Slide Shows. MD Zahidul Alam
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