Thomas Farley Microbiology Unknown Lab Report Unknown: 11C February 12, 2012 INTRODUCTION In the field of microbiology,

and medicine, scientists are able to determine a very specific genus and species of organism from a completely unknown sample by following various tests in laboratory settings. This is often an essential task, and in many medical cases is essential to determining the correct antimicrobial treatment that may save a life, or many lives. During the semester we were instructed on how to perform many of these tests and as a final project students were given an unknown micro-organism and the tools to determine exactly what it is. This is a report of the unknown I was given and the steps that I took to determine what it was. MATERIALS AND METHODS Lab 1 On the first day of this project, students randomly selected unknown samples containing the yet to be named micro-organism. I selected a test tube marked 11C, which was simple test tube containing a cloudy yellow nutrient broth. The first thing I did was prepare several streak plates using Trypticase Soy Agar. This would accomplish several things. First, it would allow me to determine what type of environment my culture would grow in. I placed plates in three different temperatures; 4C, 20C (room temp), and 37C (body temp). Secondly, I prepared a plate for an anaerobic jar to determine the oxygen requirements of the sample. The plates would also provide morphology of individual colonies, and also provide a subculture that most likely would yield better slides then a culture taken from broth. I also made a subculture in nutrient broth, and another on a slant, just to insure I had options to choose from in the future. The second thing I did was start my investigation with the Gram Stain test. In this test it can be determined if the micro-organism you are dealing with is Gram positive meaning it has a thick wall of peptidoglycan, or gram negative which would indicate the presence of a thin layer of peptidoglycan between two membranes. After following the proper steps laid out in our lab manual I was able to use the microscope to determine my sample as a Gram negative bacillus. My instructor confirmed my findings the only confirmation she would allow during the project. Lab 2 In the second unknown lab I was able to continue my testing to determine which gram negative bacillus I was working with. I retrieved all my tubes and plates from the previous labs and was able to ascertain another key clue. My sample grew in both anaerobic conditions, as well as normal

atmospheric conditions. This pointed towards it being a facultative anaerobe or a micro-organism that uses oxygen to make ATP, but can switch to fermentation if no oxygen is present. Next I determined that my sample grew well in both the 20C and 37C environments, making it a mesophile. I then determined from the flow chart handout that I should perform an Oxydase test to determine presence of cytochrome c oxidases. Using an redox indicator, I determined a negative result which then pointed me toward the Family Enterobacteriaceae and a separate flow chart. In anticipation of the new flow chart performed a lactose fermentation test by inoculating phenol red broth that had lactose sugar in it. This would show me if my sample had the ability to ferment a particular carbohydrate lactose. I also inoculated several test tubes of SIM medium, using a needle that had been dipped in my broth sample. These would at least 24 hours to show results so I would have to wait until the next lab to proceed. Lab 3 The first result I was concerned with was the Lactose Fermentation test. A positive result would have turned the phenol red, yellow. My test revealed a negative result, with the broth remaining its original red color. This result moved me to yet another flow chart, The Lactose Negative Flow Chart. The SIMs test tubes prepared in LAB 2 provided me with a lot of information. First of all, I performed an Indole test, to determine the ability of an organism to split indole from tryptophane. A positive result would turn Kovacs reagent red when added to the SIM tube. My test revealed a negative result. Looking ahead at the flow chart I was able to determine that two of the tests I would need, were already available to me this week. Since I had prepared several SIM tubes I was able to determine the results of two additional tests: the motility test, and H2S test. These were simple observations on what had happened to the SIM medium after being inoculated with my culture. The motility test measures the cultures ability to travel through the medium in an outward pattern from the initial needle stab. The H2S test can be observed at the same time, and determines the cultures ability to produce the enzyme thiosulfate reductase, which turns the SIM a dark color. Even though these observations were slightly out of order, I already knew that my unknown was positive for motility, and positive for H2S production. This was encouraging for me because based on the list of possible outcomes, I knew there was only one test left to determine my unknown; the Urease test. This test simply required me to inoculate a test tube of Urea broth and allow 24 hrs to observe the hydrolysis results. I inoculated the test tube and concluded lab 3. Lab 4 The Urea test produced a positive result, meaning no hydrolysis and allowing the medium to remain yellow due to the absence of alkaline end products. These were the results I was hoping for because my suspicions were confirmed. Unknown 11C was indeed Salmonella Typhimurium. Even

though I was certain, I preformed one last test; the endospore test. If I were correct then my sample should not produce endospores. I was relieved to find that the results of this test were consistent with my earlier determination.

RESULTS

TEST Gram Stain

Purpose To determine if the sample is gram -/+ To determine the organisms ability to thrive with and without the presence of oxygen To determine the presence of cytochrome c To determine the ability to ferment lactose determine the ability of an organism to split indole from tryptophane To determine the presence of the urese enzyme To determine the organisms ability to travel away from the stab line To determine the presence of hydrogen sulfide

Anaerobic Test

Mediums & Reagents used Crystal Violet, Grams Iodine, 95% Ethanol, Safarin Anaerobic jar, TSA plates

Observations Pink rod shapes

Results Gram negative bacillus Facultative anaerobe

Growth in both aerobic and anaerobic No color change within 20 seconds No color change after 24+ hrs. No color change after reagent added No color change after 24+ hrs. SIM medium turned almost completely purple-black SIM medium turned purple black away from the stab line

Oxidase Test

Lactose Fermentation Indole test

TSA plate, Oxidase indicator (Tetramethylphenylene) Phenol red broth with lactose SIM medium, Kovacs Reagent Urea broth SIM medium

negative

negative negative

Urese Test Motility

negative positive

H2S

SIM medium

positive

Salmonella Typhimurium:

Salmonella typhimurium is a pathogenic Gram-negative bacteria found predominately in the intestinal lumen which is why it belongs to the Salmonella Enterics group. Its toxicity is due to an outer membrane consisting largely of lipopolysaccharides (LPS) which protect the bacteria from the environment. Salmonella typhimurium causes salmonellosis and gastroenteritis in people. When the bacterial cells enter epithelial cells lining the intestine they cause irritations which temporarily damage the microvilli on the surface of the cell. This causes a rush of white blood cells into the mucosa, which throws off the ratios between absorption and secretion, leading to diarrhea. Transmission: You can get salmonellosis by eating food contaminated with S. typhimurium. The bacteria commonly come in contact with food after someone preparing the food does not properly wash hands after using the bathroom. Salmonella can also be present in the fecal matter of pets, and handling them can lead to infections if proper hygiene is not observed immediately. Signs and Symptoms: - Diarrhea -Nausea / vomiting -Abdominal pain / discomfort -dehydration Treatment: Normally the human immune system will be able to clear salmonella infections in a relatively short time (24-48hrs.) Treatments would focus more on managing the symptoms, mainly being concerned with dehydration. Anti-diarrheics such as Imodium are important to stop the loss of water and preventing dehydration.

Gram Negative Bacillus

Faculative Anarobe

Oxidase: Negative

Lactose Fermentation: Negative

Indole: Negative

Urease: Negative

Motility: Positive

H2S Production: Positive

FINAL DETERMINATION: SALMONELLA TYPHIMURIUM