Gram Stain Experiment

Decent Essays
The reason for conducting this experiment was to determine and identify a given unknown bacteria and to understand how to recognize the variance between the several types of Gram-negative bacteria. The given Gram-negative bacteria would be classified as one of the following microorganisms: Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa or Salmonella typhimurium. To confirm the Gram-negative properties of the bacteria a Gram Stain procedure was carried out and to distinguish our given unknown from the remaining five bacteria, a variety of thoroughly run tests were conducted to ensure accuracy and precision. A T Streak plate was created from the unknown bacteria and then used in the …show more content…
A Gram stain not only distinguishes between positive and negative but it also gives reputable classification in morphology, size and arrangement. Gram- negative bacteria and Gram- positive bacteria are both stained using the exact same method but G- bacteria will present itself as a red or pink color while G+ bacterial will appear purple. The Gram Stain procedure vitalizes the following 4 components: Crystal Violet, which is used as the primary stain; Gram’s Iodine, which is used as a mordant to enhance and fix the crystal violet stain; A Decolorizer, that is made up of a mixture of alcohol and acetone which allows the lipid to be removed from the cell wall making it more porous and unable to retain the crystal violet (Leboffe & Pierce, 196); Safranin, is used as the counterstain which reinstates color to the decolorized G- bacteria, causing the reddish-pink pigment. The difference in color is based on the difference in wall formation between the two bacteria. Gram Negative bacteria have a thin cell wall (8-12 nm) with high lipoprotein/lipopolysaccharide content, a thin, single layer of peptidoglycan and the presence of a perplasmic space and porins (Aryal). Gram Positive bacteria have a thick cell wall in comparison to G- bacteria (20-30 nm) with little to no lipoprotein/lipopolysaccharide content with …show more content…
The MR test assess for mixed acid production from glucose fermentation, which overcomes the phosphate buffer and lowers the pH. The MR test is also noted to produce stable acid end products. The peptone and glucose from the broth supply protein and fermentable carbohydrate while the potassium phosphate resists pH changes in the medium. To confirm mixed acid fermentation a methyl red indicator is added to the broth after a sufficient incubation time of 24 hours. Methyl red is red at pH 4.4 and yellow at pH of 6.2 and assorted shades of orange in between. A red result indicates that the bacteria are positive for mixed acid fermentation; no color change (yellow) or an orange result is either negative or inconclusive, neither of which are positive for mixed acids (Leboffe & Pierce,

Related Documents

  • Improved Essays

    Unknown Lab Report

    • 1511 Words
    • 6 Pages

    The focus of this lab was to identify an unknown organism based on its characteristics and the results from each of the tests. There will be various of test to choose from in order to identify the unknown organism, which will eliminate numerous possibilities and narrow it down to one. All the fundamental skills that we have learned and practiced in the lab will be used to perform on our unknown such as aseptic technique, microscopic examination, the use of differential media, and determining if it’s positive or negative. Performing aseptic techniques is the most crucial step that requires the utilizing of transferring, inoculating, and storing bacterial cultures and media. Aseptic technique is defined as procedures that prevent contamination…

    • 1511 Words
    • 6 Pages
    Improved Essays
  • Improved Essays

    Initially when unknown microbe #7 was gram stained and observed under the microscope, the color appeared more pink than purple, leading to the conclusion that unknown microbe #7 was gram negative. However, when completing the KOH lysis test, the microbe didn’t become sticky and remained tacky which is how a gram positive KOH test would result. It was decided to double check the KOH lysis test and the second test still yielded the same results as the first, gram positive. The conclusion of unknown microbe #7 being gram negative from the gram stain was an error.…

    • 1245 Words
    • 5 Pages
    Improved Essays
  • Improved Essays

    Gram Unknown Lab Report

    • 1105 Words
    • 4 Pages

    The gram negative bacteria included, P. vulgaris, E. coli, C. freundii, E. aerogenes, A. faecalis, and P.…

    • 1105 Words
    • 4 Pages
    Improved Essays
  • Superior Essays

    Essay On Nitrate Test

    • 1176 Words
    • 5 Pages

    During the phase and process of the bacterial unknown project, vast amounts of test were performed in order to determine an unknown species of bacteria. Within the many procedures of identifying the unknown bacteria, I was first given a tube with the unknown bacteria, from which later on I started subcultures by streaking bacteria from the original tube onto a tryptic soy agar (TSA) slant. Throughout the process of identifying the unknown, new subcultures were produced in order to use the newest bacteria to conduct the tests. Thus, this process allowed the unknown bacteria to grow and maintain. Before a series of test were conducted, first a gram stain test was performed in order to determine the unknown’s gram reaction and its cell morphology.…

    • 1176 Words
    • 5 Pages
    Superior Essays
  • Improved Essays

    After, then other biochemical tests were performed that were specific to the gram- negative bacteria to start the identification process of the unknown. The results of the gram negative tests lead to the identification of the bacteria Alcaligenes faecalis. The gram stain procedure was the first test performed by isolating a colony from the pure culture streak plate. The gram stain was followed orderly right from the referenced laboratory manual. The test resulted to be Gram- Negative bacteria from the compound microscope which were pink and rod shaped that can be seen with the 100x objective lens with the oil immersion.…

    • 1481 Words
    • 6 Pages
    Improved Essays
  • Improved Essays

    All three plates were studied, a gram stain was performed according to the referred laboratory manual noting their color and morphology, which was recorded in the journal. Gram-positive purple coccus bacteria and gram-negative rod/bacillus were identified using the microscope. The purpose of streaking on the MacConkey is to see if gram negative is lactose fermented or not and on the Mannitol salt agar if gram positive microorganism is able to ferment sugar. There were two types of bacteria that grew on the nutrient agar plates which were…

    • 416 Words
    • 2 Pages
    Improved Essays
  • Improved Essays

    Microbiology Lab Report

    • 840 Words
    • 4 Pages

    The two stains, crystal violet and safranin are used to differentiate Gram positive and gram negative cells via their cell walls. Gram Negative cells turn red due to safranin and gram positive remain purple. Through microscopic examination, oil was added onto the 400x magnification. The unknown remained purple…

    • 840 Words
    • 4 Pages
    Improved Essays
  • Great Essays

    Unknown Bacteria Essay

    • 1069 Words
    • 5 Pages

    Introduction Identifying unknown bacterium is not only vital to microbiology, but also to the medical and pharmaceutical industries in order to treat disease/infection properly. Bacteria are classified and identified to distinguish among strains and to group them by criteria of interest (1). There are several different procedures that can be utilized to identify a bacterium. This includes the oxidation test, gram stain, and catalase test. The purpose of this lab report is to show the understanding of all procedures used to identify the bacterium, and explain how the given results led to the identification of the unknown bacterium.…

    • 1069 Words
    • 5 Pages
    Great Essays
  • Superior Essays

    Gram Negative Unknown Lab Report #20 BIOL 3444-008 Giang Nguyen TA: Malini Kotak 11/05/2014 Abstract The experiment is conducted in order to identify a randomly given unknown gram-negative microorganism. By comparative analysis of biochemical tests that were performed throughout the semester to find out its characteristics and determine the identification of bacterium out of four potential unknown was given. To figure out the characteristics, T-streak was performed to isolate the sample from broth to a Tryptic Soy Agar plate, along with an additional slant in case something wrong with the original plate that can possibly affect the result.…

    • 1514 Words
    • 7 Pages
    Superior Essays
  • Improved Essays

    The properties of selective and differential media suggest that differences between the bacterial species could be used in this lab to identify these six species but their effectiveness is unknown. The efficacy of identifying a completely unidentified bacterium in a cost-sensitive manner using these test also has yet to be determined. We hypothesized that applying a combination of selective and differential media with biochemical tests would provide a presumptive identification of the first group of six unknown bacteria. We also hypothesized the identification an unknown member of the Enterobacteriaceae family referred to as unknown #53 could be definitively identified using the API 20E and other tests. To test the first hypothesis, samples of four of the six unknown bacteria were streaked onto plates of Mannitol Salt agar, MacConkey agar, and Eosin Methylene Blue agar to examine their growth on the media.…

    • 1018 Words
    • 4 Pages
    Improved Essays
  • Improved Essays

    Gram Staining Lab

    • 801 Words
    • 4 Pages

    The first test conducted was Gram staining. In the process of Gram staining, a small amount of the cultured bacteria was fixed on a slide with heat. Next crystal violet was used as the primary stain. After rinsing the primary stain, a mordant, Gram’s iodine, was added. Afterward, the slide was rinsed…

    • 801 Words
    • 4 Pages
    Improved Essays
  • Improved Essays

    Gram Stain Lab Report

    • 965 Words
    • 4 Pages

    When finding out unknown bacteria you have to go through several steps to figure out what it could be. I was given unknown bacteria number 28 and asked to run different testing to identify exactly what this bacterium is. I ran 6 test to determine my unknown. These test includes: Gram Stain, SIM, Sucrose, Citrate, MAC, and EMB. Each one of these test are different and they will give a positive or negative feedback which is how we will know which bacterium I have.…

    • 965 Words
    • 4 Pages
    Improved Essays
  • Improved Essays

    Catalase Test Lab Report

    • 654 Words
    • 3 Pages

    During the first lab period, The gram stain show that the unknown bacteria was a gram neative rod shaped microbe. After this step was established, this next part was to select the best tests and mediums to determine the unknown bacteria. This medium are use to test the metabolic characteristics of the unknown bacteria. In order to identify the unknown bacteria from other bacterias the following medium was used: MSA plate, SIM plate, nitrate reduction test Nutrient gelatin and Urease broth. For Metabolic tests, only Catalase test were performed on the unknown bacteria.…

    • 654 Words
    • 3 Pages
    Improved Essays
  • Great Essays

    Essay On Bacteria Lab Report

    • 2361 Words
    • 10 Pages

    Conclusion: The experiment has showed how some bacteria have become extremely versatile and able to live in many environments and how other have not changed much over the course of time. It has showed how use of sugar, growth based on enzymes, and ability to ferment as well as motility can help us identify multiple bacteria and distinguish them from each other. This will be useful to come in the Isolating and Identifying Bacteria project since we have already isolated and observed the bacteria the information learned in this lab gives us ways to test and observe our bacterial cultures so we can compare results back to our data and attempt to properly identify them. Real world implications of this lab show how difficult it is to obtain growth in a lab at times and how tedious it can be, but also provides a database of sorts to look back upon to help study bacteria and identify which ones cause illness, where they can be found, how to test for them to prevent illness and contamination and possible ways to fight them knowing what they need to survive and what defenses and attributes they…

    • 2361 Words
    • 10 Pages
    Great Essays
  • Improved Essays

    Gram Staining Lab Report

    • 1611 Words
    • 6 Pages

    Introduction In this lab report I use two different techniques to identify Unknown A and Unknown B bacteria’s. These techniques are gram staining and metabolic testing. I first used Gram staining to distinguished and identify the bacteria’s. Han Christian discovered gram staining in 1882, he had biopsy a patient lung that had pneumonia.…

    • 1611 Words
    • 6 Pages
    Improved Essays