Gram Negative Unknown Lab Report

The main idea of this experiment was to correctly identify the unknown bacteria. 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.Introduction 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 MethodsThe 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 test is to see whether the bacterium produces glucose from the mixed acid pathway or not. A red result means the bacteria tested positive.In the Voges-Proskauer test, I inoculated the tube with bacteria from my TSA plate and incubated the tube at 37 degrees Celsius for 3 days. After three days I placed some Barrits Reagent A and Barrits Reagent B in the test tube.

The color change of red or pink indicates a positive reaction for acetoin which tells you that the organism is a butanediol fermentor.For the Urease test, I incoluated my Urea test tube with my unkown bacteria from a TSA plate using and inoculating loop. The Urea tube was then incubated at 37 degrees Celsius for 8 days to observe for a color change. The Urea tests for the ability of a bacteria grown in urea broth produces urease. This medium contains the pH indicator phenol red.

If urease is produced the pH of the media will raise thus causing the phenol red to change from yellow to a pink color.I used an inoculating needle to stab the SIM test tube and then incubated it at 37 degrees Celsius for 24 hours. The SIM test was used to test whether an organism has the ability to reduce sulfur to hydrogen sulfide. Iron salts in the media reacts with the hydrogen sulfide to form a black precipitate called ferric sulfide.

If sulfur can be reduced than a black color will be seen in the tube. This test also sees if an organism is and indole producer. Indole producers are bacteria that produce the enzyme trytophanase which can hydrolyze tryptophan to pyruvate, ammonia and indole. To test for indole production, Kovac’s reagent is dropped into the medium.

If there is a red color than the bacteria tests positive for indole production and brown yields a negative result. The third test that the SIM test is for is motility. If the bacteria is motile then cloudiness will be seen in the medium and if not the bacteria will only be located where it was stabbed.The Citrate Test was used to test for the ability of a bacteria to utilize citrate as a sole source of carbon, these bacteria produce citrate permease which can transport citrate into the cell and make pyruvate from it. Bacteria that can utilize the citrate causes the media to become more alkaline. The indicator for this test is bromythmol blue dye which will turn from green to blue if pH is at 7.

6 and above. A blue color change yields a positive result.I inoculated the TSIA (Triple Sugar Iron Agar) Slant with bacteria from my TSA plate using an inoculating loop. The medium was then incubated at 37 degrees Celsius and checked after eight hours of incubation and again after 24 hours of incubation. This test is a differential that tests for sugar utilization, gas production and sulfur reduction.

It contains lactose, sucrose and glucose and phenol red as an indicator. No color change means that no sugars were fermented. If there is as bubble or splittin in the medium than a gas was produced and a black color signifies hydrogen sulfide production. The media has sodium thiosulfate as a reducible sulfur and ferrous sulfate as the hydrogen sulfide indicator.

The Gelatin test was solid after placing the medium in the cold room for 45 minutes thus it was negative for gelatinase. The Methyl-Red test turned red after adding methyl red to the medium indicating a positive result, thus indicating that the unknown produced mixed acids from glucose fermentation. The Voges-Prskauer test had no color change, thus indicating that the unknown was not a butanediol fermenter. My unknown remained yellow in the Urease test which means it tested negative thus indicating that the unknown was unable to produce urease. In the SIM test my unknown had negative results for sulfur reduction and motility but tested positive for indole production.

There was no black color in the medium and there wasn’t a cloudiness either however, the medium turned red when Kovac’s reagent was added indicating the ability for the unknown to produce tryptophanase. For the Citrate test, my unknown remained green which yields a negative result and the inability of the bacteria to utilize citrate as a sole source of carbon. For the TSIA slant my unknown had a yellow slant and a yellow but and it had gas bubbles in the butt thus indicating that glucose and lactose fermentation and the production of a gas. It was negative for sulfur production. All of the previous results were than compared to a gram negative unknown chart and the only bacteria that had the same results was Escherichia coli.

The unknown #3 was thus identified as E. coli. ConclusionThe identity of the unknown bacterium #3, E. coli, was supported by all of the tests that were performed for this experiment.

The search began with the identification of the bacteria as being rod shaped under the light microscope. Each test was used to eliminate the five other possible choices. The negative result of the Gelatin test eliminated Proteus mirabilis and Pseudomonas aeruginosa as possible candidates. The positive Methyl Red result further eliminated Enterbacter aerogenes. The negative Voges-Proskauer test result didn’t allow me to rule out any of the remaining candidates.

The negative Urease test result knocked Klebsiella pneumonia out as a possible candidate. The negative sulfur production of the SIM test allowed me to lastly mark Salmonella typhimurium out a possible candidate leaving only E. coli. The positive indole test was only characteristic of E. coli and the motility of the unknown was in concordance with E.

coli. Also my negative Citrate test was only characteristic of the bacteria E. coli. The TSIA slant really just helped me be sure of three other bacteria that my unknown couldn’t have been.Escherichia coli also known as E. coli is a bacterium that is prevalent in the gut of warm blooded organisms.

There are several types of E. coli that are a normal part of the human body and have many beneficial factors including the production of vitamin K2. E. coli also helps prevent harmful bacteria or pathogenic bacteria from growing inside the intestine. Most strains of E.

coli are non pathogenic however, some strains are known to cause things such as food poisoning and serious infections. E. coli has been the cause of death for many. Symptoms of an E.

coli infection include abdominal pain, diarrhea, nausea, vomiting, fever and fatigue. This pathogen is easily spreaded and has received a lot of controversy. E. coli was named for the person who discovered it, German pediatrician Theodor Escherich.I feel that this lab was very good for students such as me to see what microbiologists do on a daily basis. I really enjoyed this lab experiment and it has caused me to seek to change my major to microbiology.

I think the identification of unknown bacteria is a very important task and I really do have deep respect for the people who make this their profession. Their work helps us in so many ways such as treating illness, the cause of illness, and creating medicine.