I need my lab completed
Name: _Leslie Salgado BIOL31 Lab Lab 13: Differential & Structural Staining Techniques FCC Alignment to Labs 7, 8, 9, 10, and 11 Learning Objectives · Stain and observe microorganisms using Gram and negative stains · Observe different microorganisms using spore, capsule, and acid-fast stains. · Identify microorganisms according to their staining characteristics · Associate staining reagents with the structures they bind to or visualize General Background Information on Staining This material is adapted from “Microbiology,” lead author Nina Parker and published by OpenStax Please refer to Page 4 of the Lab 1 handout for information on smear preparation and heat fixing. Some of the information below can also be found in that handout but is reiterated here for ease of access. “In addition to fixation, staining is almost always applied to color certain features of a specimen before examining it under a light microscope. Stains, or dyes, contain salts made up of a positive ion and a negative ion. Depending on the type of dye, the positive or the negative ion may be the chromophore (the colored ion); the other, uncolored ion is called the counterion. If the chromophore is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an acidic dye. Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how the dye will interact with the specimen. In most cases, it is preferable to use a positive stain, a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background. However, there are scenarios in which it is advantageous to use a negative stain, which is absorbed by the background but not by the cells or organisms in the specimen. Negative staining produces an outline or silhouette of the organisms against a colorful background (Figure 2.32). Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly used basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains. On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin, eosin, and rose bengal. Figure 2.40 provides more detail. Some staining techniques involve the application of only one dye to the sample; others require more than one dye. In simple staining, a single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism. In contrast, differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear to be different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining. Figure 2.41 provides more detail on these differential staining techniques.” Activity 1: Performing Differential and Negative Stains (AV1-130) Part A: Gram Staining Background Written by Kate Husain for Madera Community College All cells share certain characteristics in common – they have a plasma membrane that surrounds cytoplasm, their nucleic acid blueprint is organized into some type of chromosome(s), and they have ribosomes that aid in protein synthesis. Bacterial cells are distinguished by having cell walls made from peptidoglycan, DNA organized into a large circular chromosome, and usually not have membrane-bound organelles. However, bacteria have huge structural, genetic, and metabolic variation. Many bacteria can be divided into one of two groups: 1. Gram-positive (Gram(+) or G(+)) bacteria have a thick, peptidoglycan cell wall that easily traps and retains Crystal Violet and Gram’s Iodine and one cell membrane 2. Gram-negative (Gram(-) or G(-)) bacteria have a thin, peptidoglycan cell wall enclosed between a regular cell membrane and a second outer membrane The word “Gram” comes from Hans Christian Gram, a Danish bacteriologist who developed the procedure in 1884. While he was originally trying to increase contrast of bacterial cells found in lung tissue from patients with pulmonary disease, he noticed that different cells retained different stains. Some appeared dark purple, and other appeared orange-pink or red. He refined his protocol and it became an important differential stain to identify Gram-positive bacterial cells (purple) and Gram-negative bacterial cells (pink).Figure 1 | "Gram-staining is a differential staining technique that uses a primary stain and a secondary counterstain to distinguish between gram-positive and gram-negative bacteria." (from "Microbiology" by Parker et al. for OpenStax) In this differential stain, Crystal Violet is the primary stain. The Gram-positive cells are “positive” for the primary stain at the end of the experiment, with Iodine holding the stain in place within the peptidoglycan cell wall. They appear dark purple under the microscope. The Gram-negative cells are “negative” for the primary stain and are instead counterstained using Safranin after the primary stain is washed away. They appear orange-pink or red under the microscope. Protocol (Gephardt et al, 1981, Feedback from ASMCUE participants, ASMCUE, 2005) 1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results. 2. Wash slide in a gentle and indirect stream of tap water for 2 seconds. 3. Flood slide with the mordant: Gram's iodine. Wait 1 minute. 4. Wash slide in a gentle and indirect stream of tap water for 2 seconds. 5. Flood slide with decolorizing agent. Wait 15 seconds or add drop by drop to slide until decolorizing agent running from the slide runs clear (see Comments and Tips section). 6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute. 7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper. 8. Observe the results of the staining procedure under oil immersion using a Brightfield microscope. At the completion of the Gram Stain, gram-negative bacteria will stain pink/red and gram-positive bacteria will stain blue/purple. Results Organism Total Magnification Photo or Drawing of One Field of View Gram-Positive or Gram-Negative and Additional Notes Review Questions 1. What is the purpose of a mordant and a counterstain in a differential staining protocol? 2. While Gram-staining, you are worried about adding too much decolorizer and only leave the acetone solution on for a couple of seconds. Although you thought you had a Gram-negative species, the results appear Gram-positive. What happened? Part B: Negative Staining Note: This experiment will only be done if time allows. Background Remember that in an indirect or negative stain, the background absorbs or retains the dye and the cell does not. Cellular structures such as the membrane and DNA are negatively charged. Because acidic stains are negatively charged, they are repelled by the microbial cell and are ideal for negative staining. While contrast is increased, it is the background that appears dark and the cell that remains transparent.Figure 2 | Cryptococcus neoformans using a light India ink staining preparation. This yeast causes opportunistic infections in immunocompromised patients and is particularly common and dangerous in patients with AIDS. (credit: CDC/Dr. Leanor Haley released to the public domain) Stains used in this technique – such as nigrosin(e) or India ink – are helpful for accurately measuring the cell and visualizing the cell surface. Because the negative stain procedure does not require heat-fixing, the cells are not damaged or shrunken by the application of heat. Text Protocol 1. Flame bacteriological loop until red then allow to air-cool for 30 seconds 2. Carefully drip nigrosin dye or India ink onto the sterile loop over a sink or tray 3. Gently dab the dye close to the edge of a clean slide 4. Using aseptic technique, add a loop of culture in liquid broth into the drop of dye and mix 5. Hold a second slide at a 30-45-degree angle to the slide with dye and inoculum. Place it at the front of the suspension and gently push forward to spread the dye-inoculum mixture across the slide and form a thin smear. 6. Air dry. Do not heat fix or blot. 7. Examine the slides under oil immersion. Visual Protocol Results Organism Total Magnification Photo or Drawing of One Field of View Stain Appearance and Additional Notes Review Questions 1. Why are acidic stains used in negative staining? 2. What is one benefit of negative staining? 3. Your lab partner accidentally heat-fixes the slide after you’ve prepared a negatively-stained smear. What might you expect to see when you visualize it under the microscope? You can draw or describe your response. Activity 2: Observing Differential and Structural Stains (AV1-120 and AV1-126) Note: This activity will work best if you do the “Data and Observations” portions while in AV1-120 and the background information reading and “Review Questions” portions in AV1-126. All background information and photos in this section were accessed via Microbiology by Parker et al. for OpenStax under CC attribution license v4.0. All review questions prepared by K. Husain for Madera Community College. Part A: Spore Staining Background “Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions. Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Endospore staining uses two stains to differentiate endospores from the rest of the cell. The Schaeffer-Fulton method (the most commonly used endospore-staining technique) uses heat to push the primary stain (malachite green) into the endospore. Washing with water decolorizes the cell, but the endospore retains the green stain. The cell is then counterstained pink with safranin. The resulting image reveals the shape and location of endospores, if they are present. The green endospores will appear either within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are present, then only the pink vegetative cells will be visible (Figure 2.38). Figure 2.38 A stained preparation of Bacillus subtilis showing endospores as green and the vegetative cells as pink. (credit: modification of work by American Society for Microbiology) Endospore-staining techniques are important for identifying Bacillus and Clostridium, two genera of endospore-producing bacteria that contain clinically significant species. Among others, B. anthracis (which causes anthrax) has been of particular interest because of concern that its spores could be used as a bioterrorism agent. C. difficile is a particularly important species responsible for the typically hospital-acquired infection known as “C. diff.”” Data and Observations Observe a prepared endospore stain at 1000x total magnification (using the 100x objective and immersion oil). Record the name of the bacterial genus and species: __________________________ Draw or photograph one field of view and include your results in the circle below. You should