Introduction:
This report details the steps taken and processes used to discover the identity of the unknown organism given on Tuesday, November 28th, 2017. With all of the knowledge and skills gained over the semester, in class and in lab, unknown organism 6C was able to be positively identified. The objective of these labs was to successfully utilize the tests and procedures taught during the course to correctly identify the organism and to be able to explain the reasoning behind the tests used and results found.
Procedures:
On the first day of the lab, an unknown bacteria was presented. The sample of the bacteria was labeled simply as 6C. First, the colony morphologies of the bacteria were observed. It was circular in form, raised in elevation,
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and entire in margin. The first test performed on the organism was a gram stain. A gram stain test was used to determine the thickness of the peptidoglycan in the cell wall. The organism would be gram positive if it had a thicker layer of peptidoglycan and would be gram negative if it had a thinner layer of peptidoglycan. This was determined using the gram stain. A gram stain was performed using crystal violet, Grams iodine, an alcohol wash, and safranin on a slide with a smear of the bacteria being tested. A gram positive organism would result in bacterial cells appearing purple under the microscope. A gram negative organism would result in pink bacterial cells being observed under the microscope. A test was then performed using a MacConkey agar plate. The MAC plate test was used to inhibit the growth of gram positive organisms and to identify if the organism could ferment lactose. A sample of the bacteria was inoculated onto the agar using the quadrant streaking method. Lactose fermentation was indicated on the MacConkey agar by the color of the bacterial colonies. If the organism could ferment lactose the colonies would have been pink. This pink color would have been caused by the pH drop that occured when lactose was fermented by the bacteria. A positive MAC plate would have pink bacterial colonies, whereas a negative MAC plate would have white or pale yellow colonies. Ornithine decarboxylase and indole production tests were then conducted using a MIO (Motility, Indole, Ornithine) medium. These tests were used to determine if the organism could produce indole from trophan or produce decarboxylase during dextrose fermentation. During an ornithine decarboxylase test the medium would turn from purple to yellow due to the lowering of the pH caused by the fermentation of dextrose. If the organism could decarboxylate ornithine, the pH would rise and the color of the medium would return to purple. A positive ornithine decarboxylase test will result in a purple colored medium. A negative ornithine decarboxylase test will result in a yellow colored medium. The ornithine decarboxylase and indole production tests were performed by stabbing an inoculating needle with a sample of the bacteria straight into and then straight back out of the MIO medium. In an indole production test we would add Kovacs reagent to the MIO medium and observe what color the ring at the top of the medium is. If there was a color change and a red ring was produced it would indicate the presence of indole and a positive result would be determined. Had there been no color change a negative result would have been determined due to lack of indole production. Indole is produced from tryptophan and would have indicated the presence of the enzyme tryptophanase A urease hydrolysis test was performed.
The urease hydrolysis test was used to detect the production of urease and ultimately ammonia which urease produces. The urease test was conducted by inoculating the slant of the agar. If urease was produced it would hydrolyse urea and create ammonia which would increase the pH and turn the agar hot pink. Therefore, a positive urease test would have a color change to hot pink agar, whereas a negative urease test would have no color change and the agar would remain its original color.
The last test performed to assist in determining the identity of the unknown bacteria was a citrate utilization test. This test was chosen to determine if the unknown organism could use citrate as its sole carbon and energy source. It was completed by inoculating the slant of Simmons citrate agar and allowing access to oxygen. A positive result would include a color change in the agar from green to blue, due to the utilization of citrate causing an increase in pH. A negative result would not include a color change in the agar, it would remain
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green. To determine virulence factors a blood agar test was used. The blood agar test was conducted to examine the organism's ability to lyse red blood cells. It was performed using a blood agar plate, a sample of unknown organism 6C, and the quadrant streaking method. An organism’s ability to lyse red blood cells can determine its ability to and probability of successfully infecting a host. There are three types of hemolysis alpha, beta, and gamma. Alpha hemolysis would be the partial clearing of red blood cells where the bacteria was present. Beta hemolysis would be the complete clearing of red blood cells where the bacteria was present. Gamma hemolysis would be no clearing where the bacteria was present. Another test orchestrated to determine virulence was a capsule stain.
Capsule stains are used to detect if the bacteria can produce a capsule. A capsule is a thick layer outside of the cell wall that protect the bacteria from leukocytes and helps with the bacteria's ability to invade its host. A capsule stain was performed using Congo red, acid-alcohol, and acid fusion along with a smear of the bacteria. A positive would result in the ability to see a capsule surrounding the bacteria. A negative would result in only seeing the bacteria and no capsule around it.
Results and Discussion:
The gram stain was negative. The bacterial cells observed under the microscope were pink in color from the safranin stain, but all traces of the crystal violet were washed away with the alcohol wash. The bacterial cells were observed to be small and bacillus or rod shaped.
The MAC plate test was negative. Though the color of the agar changed from pink to yellow, the color bacterial colonies was unchanged. Rather than pink colonies, there were white colonies growing on the plate.
The ornithine decarboxylase test was negative. Though the results were rendered invalid by being left to incubate over the weekend, it was determined and then confirmed that the test was negative. The bacteria could not produce decarboxylase. This caused the medium to medium to remain acidic and be yellow in
color. The indole production test was positive. A red ring was formed when the Kovacs reagent was added. This indicated that the bacteria was able to produce indole from tryptophan. Looking at the urease test it was determined that the result was negative. There was no color change to the agar. The bacteria was unable to produce urease. When examining the citrate utilization test it was unclear as to if it was positive or negative. The cap had been screwed on tight enough as to not allow for proper air flow anaerobic conditions are not conducive for this test. However, the top portion of the agar had a color change and was blue. Therefore, it was determined that the result was positive. The result of the blood agar plate test was determined to be alpha hemolysis. There was partial clearing of the red blood cells, however it was not complete. After observing the capsule stain, it was found that the result was positive. The bacteria were encapsulated. There was something unexpected however about the capsule stain. In areas where the capsule could be observed, the bacteria within the capsules were unable to be seen. This could possibly have been caused by the bacteria not being stained properly. With the tests all completed, the identity of the organism could be determined. The fact that the unknown was gram negative eliminated the gram positive organisms such as Staphylococcus aureus and Streptococcus faecalis. Taking into consideration the organisms negative MAC plate result, Citrobacter freundii, Escherichia coli, and Klebsiella pneumoniae could all be excluded. The negative ornithine decarboxylase test result narrowed the possible options down to two. It could either be Providencia stuartii or Proteus vulgaris. With the positive result of the indole production test the bacteria was able to be conclusively identified as Providencia stuartii. The negative urease test result and the positive citrate utilization test result corroborated this finding. The virulence factors of the bacteria were found to be moderate. There is a slight chance that Providencia stuartii could actually cause illness. This was determined by the alpha hemolysis result of the blood agar test and the positive capsule stain. Conclusion: The unknown bacteria that began the lab was later identified as Providencia stuartii. Through tests taught and conducted in the lab the identity of the bacteria was determined along with some of its virulence factors. Providencia stuartii is a gram negative organism that cannot ferment lactose. It cannot utilize ornithine or produce urease, but it can produce indole and it can utilize citrate as it soul source of energy and carbon. The Providencia stuartii appears to be encapsulated and capable of alpha hemolysis. Any irregularities in the properties of the bacteria are most likely the result of contamination or human error while conducting and performing tests.
Upon receiving the unknown Microorganism (M.O.) #16, I prepared a slide by cleaning and drying it. Then, using a bottle of water I placed sterile drop of water on the slide and used an inoculating loop, flame sterilized, I took a small sample of the unknown growth in my agar slant and smeared it onto the slide in a dime sized circle and then heat fixed it for ten minutes. After ten minutes had passed, I collected the ingredients needed to perform a gram stain. I got the primary stain, crystal violet, and flooded my smear for sixty seconds, and then rinsed the color off with water until the water ran clear. I then flooded the smear with the mordant, grams iodine, and let that sit on the slide for sixty seconds as well. I then rinsed the grams iodine off with water and applied alcohol to the smear to decolorize the cells; however I made sure not to over decolorize and only put enough drops on the smear till the purple ran clear. I then rinsed the slide with water and flooded the smear with safranin the counter stain and let it sit for sixty seconds and then rinsed the color off with water. I blo...
1 / 3 BIO3001 Shinhye Jeon (Heather) Professor Wahlert November 21, 2017 Identifying of Spores Belonging to the Division Pterophyta by Utilizing Phylogenetical method 1. Abstract
Each test that was used in the lab for the unknown bacteria had been performed on many different bacteria and shown that each test has different results depending on the bacteria given. The first test, the Gram stain, confirmed that the unknown bacterium was a gram negative bacilli. After performing the remainder of the tests and comparing them to the twelve negative bacteria that it could be out of it was basically a process of elimination. Basically looking at all the results and seeing which tests separated positive verses negative results the most. After reviewing all of the tests the first test that stuck out besides the gram stain was the lactose fermentation, followed by the citrate utilization test and then by the indole test. The lactose fermentation test eliminated seven of the 12 bacteria. From the five bacteria left the citrate utilization test eliminated who more of the bacteria, and last the indole test eliminated two of the three bacteria left leaving only one bacterium left. After comparing the results to the results of the 12 tests and separating which tests were positive and negative for each it was obvious that the bacteria had to be Shigella
The isolate possesses some enzymes required for hydrolytic reactions. Hydrolytic enzymes found to be secreted from the bacterium, are -amylase, casein, and PYRase. In the starch hydrolysis and casein tests, there was a zone of clearing around the bacterium, which was indicative of the secreted enzymes necessary to break down starch and casein. In the PYR test, the presence of PYRase was detected by a color change to red on the PYR disc after the addition of the PYR reagent (p-dimethylaminocinnamaldehyde). Hydrolytic enzymes for which the EI tested negative were urease, gelatinase, and DNAse. In the Urea Hydrolysis test, it was observed that the urea broth did not have a color change, indicating that there was no urease secreted to break down urea in the broth. Similarly, there was no gelatinase present to break down gelatin in the Gelatin Hydrolysis test, so the nutrient gelatin remained solid. It was concluded that the EI does not possess DNase because there was no clearing zone around the bacteria, indicating that DNA had not been
One bacterium was gram negative. It underwent four different tests. These tests were the EMB test (Eosin Mehylene Blue), the Sulfur Indole Motility (SIM) test, the Urease test, and the Simmon’s Citrate Utilization test. The EMB test checks for a bacteria’s ability to ferment lactose. This test is accomplished by placing the bacteria on Eosin Methylene Blue agar. The agar is selective for gram negative bacteria and those bacteria that can ferment lactose will have colored growth, usually a metallic green sheen.
In this lab project, the microbiology students were given 2 unknown bacteria in a mixed broth each broth being numbered. The goal of this project is to determine the species of bacteria in the broth. They had to separate and isolate the bacteria from the mixed broth and ran numerous tests to identify the unknown bacteria. The significance of identifying an unknown bacteria is in a clinical setting. Determining the exact bacteria in order to prescribe the right treatment for the patient. This project is significant for a microbiology students because it gives necessary skills to them for future careers relating to clinical and research work.
They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species.
I also inoculated a tryptic soy broth (TSB), a nutrient gelatin deep, a motility agar deep, a fluid thioglycollate medium (FTM) tube, and a TSA plate with my unknown culture. All of these inoculated media were incubated until the next class period (about 48 hours). Then when I came to class most of my inoculated tubes and my streak plate appeared to have growth. The next step I took was making a gram stain to determine the gram reaction and cellular morphology of my unknown. I used my working slant to do this, after careful examination of the gram stain, I learned that my unknown was a gram-positive bacterium. I then preceded by making a negative stain to see the size of the cells of my unknown bacteria. The cell shape was cocci and the cells occurred in clusters of tetrads. After discovering that my unknown bacteria was gram-positive cocci, I turned to page 207 of the lab manual to narrow down my options, there was only four out of the gram-positive list that were
What do bacteria need to grow? For bacteria to grow the most typical thing that they like ate a warm and moist environment, but that is not all that they like. Bacteria also like and environment with a PH that is normal or close to a human PH and bacteria also like an oxygen rich environment. The places that could be common to find bacteria in a building are a keyboard, a water fountain, and restrooms. A keyboard is a common place for bacteria because it is being touched constantly with hands when people type and hands are warm, so bacteria like them. The water fountain is another place that is common for bacteria to grow because people's warm hands are touching it and also it has water, which causes it to be moist. The last place that bacteria will we commonly found in buildings are restrooms. The bacteria like restrooms because many people are in then and also there is a lot of water in them.
In the last decade, the number of prescriptions for antibiotics has increases. Even though, antibiotics are helpful, an excess amount of antibiotics can be dangerous. Quite often antibiotics are wrongly prescribed to cure viruses when they are meant to target bacteria. Antibiotics are a type of medicine that is prone to kill microorganisms, or bacteria. By examining the PBS documentary Hunting the Nightmare Bacteria and the article “U.S. government taps GlaxoSmithKline for New Antibiotics” by Ben Hirschler as well as a few other articles can help depict the problem that is of doctors prescribing antibiotics wrongly or excessively, which can led to becoming harmful to the body.
Bacteria play a large role in our health, the environment, and most aspects of life. They can be used in beneficial ways, such as decomposing wastes, enhancing fertilizer for crops, and breaking down of substances that our bodies cannot. However, many bacteria can also be very harmful by causing disease. Understanding how to identify bacteria has numerous applications and is incredibly important for anyone planning to enter the medical field or begin a career in research. Having the background knowledge of identifying an unknown bacteria may one day aid healthcare professionals diagnose their patient with a particular bacterial infection or help researchers determine various clinical, agricultural, and numerous other uses for bacteria.
This is because the cells are normally stained with Gram's iodine solution which forms a complex with crystal violet stain that is insoluble in water. Addition of decolourizer dehydrates the peptidoglycan layer; tightening it and shrinking peptidoglycan layer of the gram positives, making the large complex not penetrate the layer. When a counter stain is added, it does not disrupt the complex i.e. purple colouration of the Gram positive cells. Therefore, in absence of Gram's iodine solution, the Gram positive cells would stain brownish red as gram negative cells, making it difficult for the identification of the unknown bacteria (Leboffe and Pierce, 2010). 13. (2 points) Control slides are missing in The Gram Stain Investigation. What are they and what is their importance in the straining
Bacterial cells, like plant cells, are surrounded by a cell wall. However, bacterial cell walls are made up of polysaccharide chains linked to amino acids, while plant cell walls are made up of cellulose, which contains no amino acids. Many bacteria secrete a slimy capsule around the outside of the cell wall. The capsule provides additional protection for the cell. Many of the bacteria that cause diseases in animals are surrounded by a capsule. The capsule prevents the white blood cells and antibodies from destroying the invading bacterium. Inside the capsule and the cell wall is the cell membrane. In aerobic bacteria, the reactions of cellular respiration take place on fingerlike infoldings of the cell membrane. Ribosomes are scattered throughout the cytoplasm, and the DNA is generally found in the center of the cell. Many bacilli and spirilla have flagella, which are used for locomotion in water. A few types of bacteria that lack flagella move by gliding on a surface. However, the mechanism of this gliding motion is unknown. Most bacteria are aerobic, they require free oxygen to carry on cellular respiration. Some bacteria, called facultatibe anaerobes can live in either the presence or absence of free oxygen. They obtain energy either by aerobic respiration when oxygen is present or by fermentation when oxygen is absent. Still other bacteria cannot live in the presence of oxygen. These are called obligate anaerobes. Such bacteria obtain energy only fermentation. Through fermentation, different groups of bacteria produce a wide variety of organic compounds. Besides ethyl alcohol and lactic acid, bacterial fermentation can produce acetic acid, acetone, butyl alcohol, glycol, butyric acid, propionic acid, and methane, the main component of natural gas. Most bacteria are heterotrophic bacteria are either saprophytes or parasites. Saprophytes feed on the remains of dead plants and animals, and ordinarily do not cause disease. They release digestive enzymes onto the organic matter. The enzymes breakdown the large food molecules into smaller molecules, which are absorbed by the bacterial cells. Parasites live on or in living organisms, and may cause disease. A few types of bacteria are Autotrophic, they can synthesize the organic nutrients they require from inorganic substances. Autotrophic bacteria are either photosynthetic or Chemosynthetic. The photosynthetic bacteria contain chlorophyll that are different from the plant chlorophyll. In bacterial photosynthesis, hydrogen is obtained by the splitting of compounds other than water.
stains on sputum’s and body fluids, and have completed a few AFB cultures. Apart from