Gram Negative Bacteria

The Gram stain was used to determine if the bacteria was gram positive or negative. A negative test shows a pink color and a positive test is a purple color. When a bacterium is negative it is because it has an outer membrane and a thin layer of peptidoglycan that is much harder to stain. A positive bacterium has a thick layer of peptidoglycan and no outer membrane that can be penetrated by crystal violet.

I began by running the starch test, which tests for the presence of starch hydrolyzing enzymes. After doing a one-line inoculation of the organism, the plate had to be incubated. Once I received an appropriate amount of growth I added the reagent iodine. The iodine turned the plate purple, formed no clear zone, and lifted the organism off of the plate, which revealed that the starch was not degraded and the enzyme was not present. The organism being lifted off the plate is unique to the bacteria Corynebacterium xerosis indicating that it was my gram positive rod. For reassurance, I ran the Phenol Red Glucose test, which tests if the organism contains various enzymes that determine if the bacteria can ferment glucose. After incubation, the broth turned orange, but this did not provide a clear positive or negative result so I ran the Nitrate Broth Reduction test. The Nitrate Broth Reduction test detects if the organism utilizes nitrate. After incubation for forty-eight hours I added Nitrate A and Nitrate B indicators. However, there was no color change indicating that the test was inconclusive. Since the test was inconclusive, I proceeded to the following step, which included adding a small amount of zinc to the broth, and this turned the broth a red color. The red color indicated that

Unknown bacteria determined to be Alcaligenes faecalis because of its morphological, physiological and metabolic properties.

This experiment was conducted to find the genus and species of an unknown bacteria prescribed by the lab teacher, which was unknown bacteria GA3 in my case. Identification of unknown bacteria techniques are used on an every day basis to figure out what type of bacteria it is and to find the best method of how to treat a patient with this bacteria (1). All five “I’s” of Microbiology were used in the testing for the unknown culture. Inoculation was used several times to put the unknown culture into agar plates or into biochemical test tubes. After Inoculation of these tubes or plates, they always were placed into the incubator for further growth and development. Isolation was used to make sure we got the correct bacteria we were testing for. After each further isolation, we gram stained the culture and inspected the culture under a microscope to further help in the identification process of the unknown bacteria. Multiple tests were done on the unknown culture to make sure we were confident in what kind of bacteria the unknown was.

The first step to identifying the unknown bacteria residing on the blood agar plate sent in from Khokana was to do a Gram stain on it. This is an important first step because it dictates further testing that will be necessary to arrive at a final conclusive result. Viewing the fixed and stained slide under the microscope revealed round chains of bacteria in a purple color signaling Gram-positive streptococci. A catalase test was performed with no bubbling present indicating a negative result. This further confirmed the shape and arrangement seen under microscopy. With this mind, the coagulase test was not done, as it would be of no use since that specifies for staphylococcus, specifically for Staphylococcus aureus. For streptococcus, an examination of hemolysis was necessary at this point. Shifting attention back to the original blood agar plate, gamma hemolysis was noted, thus narrowing the field down to two choices left. The unknown bacteria was either Streptococcus bovis or Entercoccus faecalis. This also means the Optochin and Bacitracin sensitivity tests would not be needed as those pinpoint alpha- and

Clostridium difficile is a Gram-positive, spore-forming, rod-shaped bacillus that is renowned for being the leading cause of hospital-acquired diarrhea in adult patients. C. difficile is present as normal intestinal flora within 3% to 5% of healthy people2, while its spores are ubiquitous in the environment, especially in hospital settings. It grows at an optimal temperature and pH of 37ºC and 6.5–7.5 respectively.1 It is an obligate anaerobic as it thrives in the absence of oxygen. It is highly motile with the presence of peritrichous flagella, which are evenly spread out along its surface. As briefly mentioned above, this evolving pathogen produces endospores. The bacterium produces dormant spores, which are extremely hardy and resistant to antibiotics, the host’s innate immune system, and once shed into the environment through the host’s feces, they are resistant to unfavorable aerobic conditions3 as well as several types of bleach-free disinfectants, which are commonly used in hospitals.3 The spores will germinate under the favorable conditions of the intestinal tract, resulting in the multiplication of vegetative cells, colonizing in the gastrointestinal tract. The vegetative cells release two powerful exotoxins upon adherence to the epithelial cells of the GI tract. Pathogenic strains of C. difficile produce two exotoxins: toxin A and toxin B. Toxin A is an enterotoxin that causes fluid excretion, resulting in fluid accumulation and watery diarrhea. Toxin B is a potent

The main idea of this experiment was to correctly identify the unknown bacteria, #3. 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

This lab experiment was done for the purpose of learning how to determine a gram negative bacterium based on multiple tests learned throughout the semester. My gram negative unknown bacterium given to me was Salmonella typhimurium based off of the following tests; Triple Sugar Iron Agar (TSIA), Sulfate Indole Motility (SIM), Methyl Red (MR), Voges-Proskaur (VP), Citrate, Urea Hydrolysis, and Gelatin Hydrolysis. Each test performed gives results such as motility, acid production, fermentation, carbon requirements, or detection of certain coenzymes. With a process of elimination, I determined which bacteria it was not and which bacterium I had, S. typhimurium. The expectation was to master the techniques for each test and utilize the results to determine the unknown bacterium I was given within a two-week period.

The purpose of this lab was to identify an unknown microorganism using lab techniques. The importance of identifying microorganisms is essential to the survival of humans, expansion of modern day medicine and improvement of quality of life. In 1884, Hans Christian Gram designed a differential staining technique to identify bacteria that would change the future of microbiology. He give rise to a staining process, known as the Gram stain to differentiate microorganisms into two groups between positive and negative gram staining microorganisms. The Gram stain is essential in a lab technique as it distinguishes the cells based on the physical properties of the individual cell walls, and is almost always the first test preformed to differentiate a microorganism. The identification of weather a microorganism is gram positive or negative can revel the bacteria’s virulence, cell wall structure, resistance to antibiotics, resistance to physical disruptions and so much more. In order to identify the unknown provided, unknown #27, the Gram stain was the first test preformed. After discovering that the unknown bacterium was indefinitely a gram positive microorganism, the vast possibilities were narrowed down. However, In order to more definitively identify the unknown, the next step was to preform biochemical tests. A biochemical test identifies metabolic

Carbohydrate testing utilizes the different ways that bacteria metabolize different sugars by inoculating different broths with the test bacteria and seeing if there is a change in acidity and/or if any gases are produced. For glucose testing, we would check to see if gas is produced and if the ph of the broth solution drops for a positive test result. For lactose, we check to see if the ph drops or becomes more acidic. For sucrose we check the same was as for lactose but use a sucrose solution instead. A negative result would mean that the solution contained a base/alkali ph.

When checking the results, H2S was negative, there were no black precipitants in the tube, and the organism was motile. After recording those results, I went to the hood to add Kovoc’s reagent to the SIMs and waited for about 30 minutes before observing to get the results, and it came out to be negative for indole as well. At this point, only one organism matched every result that I received during all my tests, I decided to do my confirmation test. I did Methyl Red and Voges-Proskauer tests for my confirmation test. After inoculating the MRVP broth and incubating it at 37oC for 120 hours, I took my separated samples to the hood and added in the Methyl Red in one tube and reagent A then B in the other tube, I mixed it and checked in on both tubes after every 10 minutes. About 45 minutes later it was clear what the results were, negative for methyl red and positive for Voges-Proskauer. That confirmed that my unknown O17 was Enterobacter

In the world of microbiology it is vitally important to be able to discern the identities of microorganisms. Not only is it important in a lab setting but as well as in healthcare in general. Properly identify what strain of bacteria a person has will aid in the proper medicine and dose given. Throughout the semester we have learned about different types of bacteria and certain test that can clearly identify them. The purpose of this lab report is to identify a Gram-positive or Gram-negative bacterium. Using all the knowledge of procedures and lab techniques identify the unknown and discuss all the tests you performed.

The bases of this experiment was to discover the identify of the unknown from three possible specimens: Klebsiella pneumonia, Escherichia coli, and Enterobacter aerogenes. Utilizaing the T streak technique, the bacteria was isolated into pure colonies for further study. The Gram Stain method was used to identity the morhphology of the bacteria such as the shape and whether the bacteria was Gram positive or Gram negative. Biochemical test were also used to help identify the unknown bacteria. The biochemical test used was the Triple Sugar Iron Agar, Sulfur Indole Motility test, Methyl Red test, Voges-Proskauer test, Citrate test, Urease test, and the Gelatin test. After observing the morphology of the bacteria using the Gram Stain method and utilizing all the possible biochemical test, the bacteria was identified to be Enterobacter aerogenes.

Shigella is a Gram-negative organism. Transmission occurs via the fecal-oral route or through person to person contact. The symptoms usually appear 1 to 2 days after ingestion and lasted up to 7 days. Shigella is characterized by “bloody diarrhea; fever; and stomach cramps” (Friis & Dworkin, 2010, p 110). Because of their lower infectious doses many people with Shigella are only mildly ill therefore contact with other persons can further transmit the infection (Friis & Dworkin, 2010, p 110). The spread of Shigella from an infected person to another can be avoided by frequently washing hands with soap and warm water. Hand-washing among children should have been supervised to make sure it was done correctly, especially after toileting and activities.

After multiple differential tests, it was concluded that unknown #20 was Citrobacter freundii. After conducting the Gram stain to ascertain that the unknown bacterium was a Gram negative bacilli, the species were inoculated in multiple biochemical tests. The tests that led to the conclusion of a Gram Negative bacteria were Oxidase, lactose fermentation, MR, VP, Indole, and H2S production. For the oxidase test, there were two categories one was for positive and negative results of the test. The positive result had three different types of bacteria while the negative result was the Enterobacteriaceae family. If the result of the test was positive, then there would have been color reaction. However after the test was performed in the oxidase test it showed no color reaction which indicates that the test was negative fitting the Enterobacteriaceae classification. Following was lactose fermentation test to analyze if the bacterium was capable of fermenting sugar. If the test was negative the tube was stayed the phenol red and no gas would have been collected by the Durham tube. However, after being incubated with bacteria, it was observed that the phenol red turned yellow and gas was collected in the inverted tube, signifying the bacteria grew. The changing of the color demonstrated that the pH dropped and that the bacteria is able to ferment sugar. Due to the positive result of the test therefore it narrowed down to a small amount of bacteria. To further narrow the list of