Gram Staining
Formal Lab Report
Miranda King
Intro Microbiology
Dr. Hendrickson
13 September 2017
Purpose
For the purpose of the gram staining experiment was to learn this specific type of staining technique, as well as learn how distinguish the differences between Gram positive and Gram negative stains. This technique uses crystal violet stain, Gram’s Iodine, Ethyl Alcohol, and Safranin. These dyes are used in order to distinguish between the different types of cells. For example, Gram positive staining will result in a purple color, and Gram negative staining will result in a red or pinkish color (University of Central Oklahoma department of biology, 2016). When a bacteria results in a purple stain it is categorized as a gram-positive stain because the alcohol that was used to decolorize the bacteria was not successful. Its’ cell wall is made with a thick layer of peptidoglycan and holds the crystal violet color (Case, Funke, & Tortora). When a bacteria results in a red or pinkish color, this means that it loses the crystal violet dye color after being decolorized and keeps the Safranin dye. It would then become categorized as a gram-negative stain (Case, Funke, & Tortora). A gram-negative bacteria has a cell wall made of a thin layer of peptidoglycan and a thick layer of lipopolysaccharides thus holding onto the red dye (Case, Funke, & Tortora). Bacteria have different shapes and these shapes determine what family they belong in. There are
Escherichia coli cells would be pink following a gram stain. E. coli is a gram negative cell due to its thin peptidoglycan layer and its outer membrane structure. In the gram staining process, the outer membrane does not play a role in staining but the peptidoglycan
I learned that anaerobic is an organism or tissue that is living in the absence of air or oxygen while aerobic is involves the organism or tissue receiving and requiring air. Furthermore I learned about the anaerobic cellular respiration that uses an electron acceptor rather than oxygen to complete metabolism using electron transport-based chemiosmosis. Also in this reading I learned about fermentation which is an anaerobic process in which energy can be released from glucose even though oxygen is not available.
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.
This experiment was given to us to utilized previous knowledge learned throughout the semester to identify a gram negative unknown bacterium. We had to first learn the difference between a gram negative and a gram positive organism. We started off with doing gram stains to determine whether it was positive or negative. Based on the gram stains, a gram positive stains purple and a gram negative stains pink. A gram positive stains purple because the cell walls is made of a thick peptidoglycan layer and doesn’t
The next step of the project included preparing a Gram stain to discover the cell shape, arrangement, and if the bacteria is gram positive or
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
The chart below shows the biochemical tests of the gram stain below. Test performed Purpose Materials used Observation Results Gram stain test Gram reaction to specimen Crystal violet, iodine, alcohol safranin Pink/purple colored Gram positive cocci/ gram negative rods After the gram stain showed the specimen as Gram positive cocci, and Gram negative rods, more tests are necessary. Test performed Results Enterotube II Gram negative rod: Citrobacter
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.
The first result of importance was the result of the Gram stain. The observations of the unknown bacteria from the slant culture after Gram staining showed that the unknown bacteria were Gram negative bacilli (Image 1). After determining the unknown bacteria was Gram negative, an oxidase test was conducted on a sample from the slant culture. The cotton swap with the sample of bacteria did not change color when the oxidase reagent was applied, thus providing a negative result. With a negative oxidase test, further tests were conducted to determine various characteristics of the unknown bacteria. A MR-VP broth was inoculated with a sample from a slant culture of unknown bacteria. After incubation, the methyl red reagent was added to the broth, and the broth turned red, providing a positive result (Image 2). An EMB agar streak plate was inoculated with a sample from a slant culture of the unknown bacteria, and after incubation, growth was found on the plate, providing a positive result (Image 3). A Citrate agar slant was inoculated, and after incubation, growth was found on the media, providing a positive result (Image 4). A Urea agar slant was inoculated, and after incubation, the agar had changed from a peach color to a bright pink color, providing a positive result (Image 5). Using the flowchart (Figure 1) developed from the Table of Expected Results, the lab partners started at the oxidase test. Given the negative result of the oxidase test, the flowchart is
If feeding efficiency and reproduction have a direct correlation, and a population started with equal proportions of individuals with each of three feeding types, metal spoon, metal knife, and plastic fork, the frequency of the population with metal spoons as their feeding structure will increase in the next generation. While the frequency of metal knifes and plastic forks will decrease. Furthermore, since the organisms with the metal spoon feeding structure have a higher fitness level, this population will evolve by natural selection to a point where the metal spoon phenotype will be in abundant. While the organisms with metal knifes and plastic forks phenotypes will decrease in frequency due to the lack of reproduction. Eventually, if this population persist overtime, most of the organisms, if not all, will have the metal spoon phenotype, while very few, if not any, will have the metal knife or the plastic fork phenotype.
I know this because of the Gram stain that I performed on the first day of the unknown project. The Gram stain dyes the cell either dark purple, which is a Gram positive, or pink, which is
An association between enzyme production, gene copy number, and gene evolution was explored by conducting analysis of the salivary amylase enzyme, AMY1A gene copy number, and the ancestral starch consumption in Homo Sapiens (Tracey 2017, p.22). It was hypothesized that the relative amount of starch consumption was very high for my personal ancestral diet, thus my AMY1 diploid gene copy number in my genome and salivary amylase concentration would be significantly higher than the population mean. With a population of 28 subjects (n=28), individual saliva samples were collected and compared to a calibration curve to determine the approximate amylase concentration by analyzing absorbance values. Individual samples of buccal cheek cells were
Crystal violet stains the cell of the bacteria and is placed over the heat fixed slide for one minute then rinsed with H2O. The mordant that binds the stain is gram’s iodine and is recommended to be present on the slide for one minute. Alcohol is used as the decolorizer and is dripped over the recently rinsed slide until the slide runs slightly blue. If the bacterium retains the crystal violet it is due to the thicker peptidoglycan in the cell wall (gram-positive). The bacteria will then be stained with the counterstain safranin for 45 seconds. When crystal violet is not retained in the cell wall of the bacterium it will stain pink (gram-negative). A gram stain was immediately performed with the recently inoculated streak plate. An inoculating loop was used to gather some of the bacteria from an isolated colony. The loop was placed on a new slide and the transferred bacterium to the slide. The slide was dried, heat-fixed, and the gram stain process just described was completed. After microscopic analysis, it was determined the first unknown bacteria was a gram-positive cocci. The second unknown was determined to be gram-negative cocci. With this information a set group of test decided and performed based on the Unknown Bacteria Flow Chart as provided in the lab manual (Pg.) these test and the results are shown in record on the following pages. These are separated for the gram positive and the gram negative
Escherichia is a genus of aerobic gram-negative rod-shaped bacteria of the family Enterobacteriaceae that form acid and gas on many carbohydrates, such as dextrose and lactose, but not acetone, which include occasional pathogenic forms, including some strains of E. coli which are normally present in the human intestine as well as other forms which typically occur in soil and water (Webster). Escherichia coli is a gram-negative bacilli that rarely varies in shape and size and when stained often resemble safety pins because the ends of some bacilli stain more densely than does the middle; which is a characteristic called bipolar staining which is common in enteric gram-negative bacilli (ASM). Gram negative cells have a thin cell wall layer and will stain red to pink. The staining process is the same as Gram positive, requiring four steps: applying a primary stain, adding a mordant, then rapid decolorization and completing with a counter stain. Applying the alcohol for decolorization dissolves the outer membrane and leaves small holes in the thin peptidoglycan layer through which the crystal violet-iodine diffuse. The gram-negative bacteria is colorless after the decolorization; therefore adding safranin
Gram staining is a technique used to determine if the bacteria is Gram positive or Gram- negative. Gram staining procedure uses crystal violet stain, iodine moderator, alcohol decolorizer and safarin counter stain. In Gram- negative bacteria the primary stain will be washed out with the decolorizer and it will be stained with the counterstain. Whereas in Gram-positive bacteria the primary stain will not leave the cell wall. This difference comes from difference in the structure of the cell wall that retains the stain.