Environmental Unknown Experiment for Microbiology
The information contained on this site is correct to my knowledge. I obtained all information from my personal research and testing. Information from this site may be borrowed, but please site your references correctly. Incorrectly sited references constitute PLAGIARISM!!!!!!! There are various laws and regulations that govern the use of another person's work for personal gratification.
James Arnold
Microbiology
November 19, 1999
Environmental Unknown Lab Experiment
The purpose of this experiment was to get the class to use the laboratory techniques used in the lab setting to figure out the identity of each of our individual bacterial specimen. This experiment would require each student to have an adequate understanding for the safety measures and laboratory techniques used each week in the laboratory. All of the tests run each week would have to be run on our unknown bacterial specimen and applied to a flow chart of the possible identity of the bacteria.
The procedure for the experiment was carefully mapped out for us on a flow chart that we followed during our exploration for the identity. The experiment started when we were given two petri dishes and two sterile cotton swabs and told to take a bacterial sample from anywhere we wanted. I went got my unknown sample from out of my sink from under the drain plug. After collecting the bacteria, we were going to have to isolate the bacteria from the other bacteria that may be growing on the dish. For this to occur, we used the technique called streak for isolation. This technique is achieved by first finding a colony on the plate that is easy to sample with the inoculation loop. After this is achieved, the bacteria are streaked on a new petri dish in three directions. The first direction is for a lawn growth. The next streak is done in one direction starting from the end point of the lawn and away at a right angle, flaming the loop between each streak from the lawn. The third streak is done from the location of the second streak. Again, it is done in one direction starting from the end area of the second streak and in one direction away from the area at a right angle. This last streak is the point at where you are to look in order to see isolated colonies of bacteria after 24 hours of growth. The end product of the streak before incubation should be a figure similar to a three-sided square.
After growing the bacteria for 24 hours, isolated colonies should be seen on the petri dish. One of these colonies must me selected and inoculated using the inoculating loop into a nutrient broth for more growth. From the bacterial growth in the nutrient broth or a fresh growth of bacteria (24-hour culture) a gram stain can be conducted. The gram stain is a test that determines whether bacteria are gram positive or gram negative. To do this, the bacteria is either taken from the broth or is taken from the petri dish and the following procedure is conducted.
1. The bacteria are smeared on a slide and allowed to dry.
2. The bacteria is heat fixed to the slide
3. Crystal violet pigment is added to the smear for 1 minute
4. Wash the pigment off with distilled water
5. Apply Gram’s Iodine for 1 minute
6. Wash iodine off with distilled water
7. Decolorize the smear with 95% ethyl alcohol drop by drop until it runs off clear
8. Wash off alcohol with distilled water
9. Counterstain with Safranin for 45 seconds
10. Wash off Safranin with distilled water
11. Blot dry with Bibulous paper
Following the staining procedure the slide is to then be viewed under a light microscope under oil immersion in order to determine the shape of the bacteria and its gramness. My unknown was found to be bacillus shaped and be gram-negative as denoted by its pink color under the microscope. Gram positive cells appear as blue or purple. As following the flow chart, the next test that I must conduct is the Oxidase test. This test determines whether a bacterial genera uses cytochrome oxidase to catalyze the reduction of cytochrome by molecular oxygen to produce water and hydrogen peroxide. Use of this enzyme is exhibited in both aerobic bacteria and facultative anaerobes. This test will aid in differentiating between the Neisseria and the Pseudomonas, which are oxidase-positive and the Enterobacteriaceae, which are oxidase-negative. At this point, my hypothesis is that my unknown is a member of the Pseudomonas genera as determined by other tests that I have run to include the Triple Sugar-Iron test, gas production screening, the Hydrogen Sulfide Test, and gelatin hydrolysis screening.
The Triple Sugar-Iron test is used to differentiate among the different groups or genera of the Enterobacteriaceae, which are gram-negative bacilli capable of fermenting glucose with the production of acid and to distinguish the Enterobacteriaceae from other gram-negative intestinal bacilli. This test is outlined in the lab manual as Exercise 23 beginning on page 143. The agar used contains the sugars sucrose (1%), lactose (1%), and glucose (.1%). The acid-base indicator in the agar is phenol red, which changes from red to yellow in the presence of acids. The inoculation method is the stab and streak technique used on TSI slants that involves using the stab to inoculate bacteria down to the bottom of the tube and then making a streak of the bacteria on the slanted portion of the agar. The result of my unknown was that the butt of the tube turned an orange-red color and the slant remained red meaning that some carbohydrate fermentation took place followed by catabolization of peptones, forming ammonia. The alkaline ammonia caused the butt to become orange-red. From this test, I also found that no gas was produced during fermentation. The next test conducted was the Hydrogen sulfide test.
The Hydrogen Sulfide test, as described in the lab manual on page 157 is used to determine the ability of microorganisms to produce hydrogen sulfide from substrates such as sulfur-containing amino acids or inorganic sulfur compounds. The substrates used in this test are peptone and sodium thiosulfate, which are mixed into the SIM medium. The hydrogen sulfide indicator is ferrous sulfate, which turns black when hydrogen sulfide is present. The procedure used is the stab technique. The unknown bacterium is inoculated into the tube of semisolid agar using a flamed stab. After 24 hours, the tube was checked and it was found that the bacterium did not produce hydrogen sulfide because the tube was its original light brown color. We found that the bacterium was both non-hydrogen sulfide forming and it was non-motile because it didn’t spread from the inoculation line that went down into the tube. The next test conducted was the gelatin hydrolysis test.
The Gelatin hydrolysis test, as illustrated in the lab manual on page 132, is used to determine whether a bacteria has the capability of producing the proteolytic enzyme gelatinase, which acts to hydrolyze this protein to amino acids. At normal conditions, the gelatin is liquid at 25C and is semisolid at temperatures lower than 25C. With the enzyme gelatinase present, the gelatin will stay liquid even at temperatures below 4C because the gelatin protein will have been hydrolyzed. In this test, the gelatin agar is prepared and allowed to cool to below 25C so that it will be solid. The bacteria are then inoculated using the stab method down into the tube of cooled gelatin. The tube is left to incubate for 48 hours at 37C and then is placed in a refrigerator to cool it to 4C for 30 minutes. After 30 minutes, the tube is examined to see if it is solid. If the tube is solid, the bacteria inside the tube did not hydrolyze the gelatin. If the tube is liquid, the bacteria have the enzyme gelatinase and can hydrolyze gelatin, just as my unknown. It is positive for gelatin hydrolysis and that strengthens my hypothesis that the bacteria are of the Pseudomonas genera. According to the flow chart, if the oxidase test proves positive for my unknown, it will be of the Pseudomonas genera. I won’t be able to tell until the results of the oxidase test have been reviewed.
The oxidase test is designed to distinguish between groups of bacteria based on cytochrome oxidase activity as illustrated in the lab manual on page 179. As said previously, the oxidase test will aid in differentiating between the Neisseria and the Pseudomonas that are gram-negative and oxidase positive. The oxidase test was conducted on the unknown bacteria and was found to be negative. This means that he unknown cannot be either Pseudomonas or Neisseria and that it is a fermentative facultative anaerobe. This means that my choices are now Klebsiella or Shigella. The citrate test has to be conducted to differentiate the two.
The citrate utilization test, as described on page 150 of the lab manual, is designed to differentiate among enteric organisms on the basis of their ability to ferment citrate as a sole carbon source. The principle behind this test is that some microorganisms have the ability to ferment citrate in an environment that lacks fermentable lactose or glucose to achieve carbon for the Krebs cycle. In order to ferment citrate, the bacteria must have the enzyme citrase. With this enzyme, citrate is broken down into acetate and oxalacetic acid, which are then converted into pyruvic acid and carbon dioxide. The carbon dioxide produced combines with sodium and water in the agar and forms sodium carbonate, which is an alkaline product. This changes the agar color from green to Prussian blue. Citrate-positive bacteria are identified by both the presence of growth on the surface of the slant and by the blue color of the slant. Citrate-negative bacteria will show no growth on the slant and the color will stay green. My bacterial unknown proved not to grow on the slant and the color stayed green meaning that my unknown was citrate-negative. This must mean that my unknown is a member of the Shigella genus.
The Shigella genus is known for causing foodborne diseases that often are transmitted through the oral-fecal contact. This means that personal hygiene and proper food preparation is essential in combating the effects of he diseases caused by this genus. The main disease caused by Shigella is dysentery. The symptoms are bloody diarrhea, vomiting, dehydration, fever, nausea, and cramping. This disease is more prevalent in areas where population is high and where tropical conditions exist. This makes under developed countries at a high risk because of substandard sanitation. This disease will kill in its first 24-48 hours if not controlled due to rapid dehydration. The general rule is that if the victim survives the first 24 hours with the disease, he stands an 85% chance of surviving the disease. Dysentery usually kills its victims in the first 24-48 hours because it grows so fast that the body cannot react to the rapidly increasing number of bacteria in the blood. After this first stage, the body can then effectively react against the disease both on its own and because its lost nutrients will be replenished through IV.
My Favorite Web Sites
Angelfire - Free Home Pages
My Main Page
Email: arnoldj@citadel.edu