There are many types of microorganisms and ways to treat each one. Knowing the differences of each is vital to treat a patient correctly. The purpose of this report is to explain the process and steps used to identify a certain microorganism referred to as the unknown.
Materials and Methods
A test tube with an unknown microorganism will be retrieved. Once the test tube is retrieved, a steak for isolation will be completed in order to produce isolated colonies of the organism on an agar plate. The unknown test tube with the bacteria is flame sterilized using a Bunsen burner. Once the bacterium test tube has been flame sterilized, a flame sterilized inoculating loop will be used to gather some of the bacteria from the test tube. The bacteria will be spread onto a plate using the streak for isolation method which divides the bacteria into four sections. The agar plate will then be incubated at 37 degrees. After the streak for isolation is completed, a gram stain will be done. The gram stain is when a decolonization step occurs between the application of two basic stains which will determine if the bacterium is gram-negative or gram-positive.
Using a flame sterilized inoculating loop, obtain a small amount of the unknown bacteria from the flame sterilized glass test tube. The bacteria will be added to a clean glass slide with a small drop of sterile water. The glass slide will need to be completely dry before it is heat fixed. Once the glass slide is dry, it will then be heat fixed using the Bunsen burner flame. Crystal Violet stain will then be added for 60 seconds and then rinsed with sterile water. Grams Iodine will be added for 60 seconds and then rinsed with sterile water followed by Ethanol but not for more than 10 seconds. The Ethanol will be rinsed from the slide with sterile water and Sarasin will be added to the slide for 60 seconds. After the time is completed, the slide will be rinsed with sterile water followed by blotting it with bibulous paper. The slide can then be viewed under a microscope.
The 100x magnification will be used and oil will be added to the slide for the oil immersion. Once the Gram Stain is complete a Triple Sugar Iron test will be used to test the microorganism’s ability to ferment sugars and to produce hydrogen sulfide (Leboffe & Burton 329). Add the bacteria from the unknown test tube to the Triple Sugar Iron agar by stabbing a flame sterilized inoculating needle into the butt of the agar. After stabbing the butt, the slant will then be streaked. The tube will then be incubated aerobically. The next test to be completed will be the SIM medium test. The SIM medium test is used for determination of sulfur reduction, indole production from trophan and motility (Leboffe & Burton 323). Using a flame sterilized inoculating needle, bacteria will be collected from the unknown test tube.
The needle will be stabbed into the butt of the agar about 1 cm from the bottom. The inoculating needle will need to be removed from the agar along the stab line. The tube will be incubated aerobically. After the incubation process is complete, Adding Kovac’s reagent to the test tube will either produce a red color in the upper layer or not to indicate indole production. The next test to be completed will be the Phenylalanine Deaminase Test (PDT). Organisms that produce phenylalanine deminase can be identified by their ability to remove the amine group from the amino acid phenylalanine. The slant of the agar will be streaked with the bacteria using a flame sterilized inoculating loop. The tube will then be incubated aerobically.
The tests were performed using the unknown test tube labeled number one. The results from the streak for isolation showed bacterial growth, clusters of cells and colonies.
The gram staining procedure showed multiple pink rods under the microscope. Once the oil was added the rods and color became more visible.
Completing the Triple Sugar Iron Test showed a yellow butt with a yellow slant and the production of gas.
The SIM medium test showed the original color of the agar. Once the Kovac’s reagent was added, a slight red color appeared at the top of the agar.
The Phenylalanine Deminase Test showed results of the original color of the agar along with an orange color towards the bottom of the slant.
Once unknown number one was chosen, the first test to be completed would be the streak for isolation. The streak for isolation was chosen to see the formation of the different colonies and to see if the bacteria would cause growth on the plate. After observing the plate, it could be seen that the unknown had growth and formation of colonies. The Gram stain was chosen next to determine if the unknown was gram-negative or gram-positive. After looking at the gram stain under the microscope with the oil immersion it could be determined that the unknown was gram-negative due to the fact that the bacteria was pink in color and rod shaped. After determining the unknown was gram-negative, other bacteria’s were able to be eliminated from the options. The Triple Sugar Iron Test was chosen next because the unknown could be tested for glucose fermentation, lactose fermentation, sucrose fermentation and sulfur reduction.
If the organism is able to ferment glucose and lactose and/or sucrose it will turn the medium yellow throughout (Leboffe & Burton 329). When the Triple Sugar Iron Test was complete, it showed a yellow butt, yellow slant and gas production. This determined that the unknown ferments glucose and one or both of the other sugars. The gas was produced by fermentation. The color change in the agar brought the unknown down to two options of unknowns, Escherichia coli or Hafnia alvei. The SIM medium test was chosen next to see sulfur reduction, indole production from tryptophan and motility. After the Kovac’s reagent was added to the TSI test tube, red color change could be seen at the top which indicated a positive reaction in the presence of tryptophanase.
Since there was no other color change in the test tube, it could be determined that there was not any sulfur present. Since Escherichia coli is positive for the production of tryptophan and hafnia alvei is not it could be determined that the unknown was Escherichia coli. The Phenylalanine Deaminase Test was completed just to be sure the results checked out to be Escherichia coli. The results showed an orange color towards the bottom of the slant. This determined that it was negative for the ability to remove the amine group from the phenylalanine. Escherichia coli checked out for the negative reaction towards the PDT test. After reviewing all the results from the different tests the unknown was determined as Escherichia coli.
Escherichia coli is a bacterium that usually lives in the intestines of humans and animals (Centers for Disease Control and Prevention). Most types of Escherichia coli are harmless and are actually a part of a healthy human intestinal tract but some are pathogenic. It can be transmitted multiple ways; fecal-oral route, fecal contamination of water, food or fomites, poor sanitation and hygiene (General Microbiology Fact Sheet). A person can show signs and symptoms of Escherichia coli which include fever, bloody diarrhea, may result in hypotension with severe toxemia. The incubation period is 12-72 hours. Knowing the steps and process of finding an unknown is helpful when it comes to the different types of bacteria’s and which are harmful to the body and how to treat them.
Leboffe, Michael J., and Burton E. Pierce. Microbiology: Laboratory Theory and Application. 2nd ed. Englewood, Colo.: Morton Pub., 2006. 329. Print. Web. 20 Nov. 2014. . “E. Coli (Escherichia Coli).” Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 22 May 2014. Web. 20 Nov. 2014. .
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