Examine all three slides under oil immersion and record your results on your worksheet. Repeat this procedure to make a slide of S. A quick summary of the Gram stain thats pertinent to. Allow the slides to air dry on the counter. The technique essentially made bacteria more visible to scientists when examined under the microscope. Use a second slide, held at a 45-degree angle to smear across your slide.ġ2. A Gram stain of a mixture of Staphylococcus aureus (Gram-positive purple cocci) and Escherichia coli (Gram-negative red rods). Also, shown are two typical bacterial cell shapes: the bacilli (or rods) and the cocci (or spheres). Aseptically transfer one loopful of your NEGATIVE STAIN MIX bacteria into the drop of nigrosin and mix gently.ġ1. The right column shows how Gram-positive bacteria react. Add a small drop of nigrosin to the slide.ġ0. If you need to, step outside and watch this video to make sure you understand how to do the procedure: (you can also google “negative stain video”)ĩ. The glass of the slide will stain, but the bacterial cells will not.Ĩ. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. Utilizing gram stain allows microbiologists to look for characteristic violet (Gram +) or red/pink (Gram -) staining patterns when they examine the organisms under a microscope. Nigrosin is an acidic stain which becomes negatively charged. Gram stains are useful for quickly identifying if bacteria are gram positive or gram negative, based on the staining patterns of their cellular walls. An advantage of using this method is that prior fixation by heat is not needed, so the organisms are seen in more lifelike shapes. The shapes and sizes of the organisms are seen as color-free outlines against the dark background. Nigrosin is a simple and indirect stain used for determining bacterial morphology. gram-negative Testing Types Pathogenic bacteria Treatment Summary Gram-positive bacteria are bacteria with thick cell walls. Similarly, Hemophilus spp., Legionella app, and some anaerobic bacteria stain poorly with safranin.\) Some laboratories use safranin as a counterstain however, basic fuchsin stains gram-negative organisms more intensely than safranin. The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative bacteria pink color for easier identification. The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria. In contrast, solvent dehydrates the gram-positive cell walls with the closure of pores preventing diffusion of violet-iodine complex, and thus, bacteria remain stained. With the dissolution of the lipid layer, gram negatives lose the primary stain. Initially, all bacteria take up crystal violet dye however, with the use of solvent, the lipid layer from gram-negative organisms is dissolved. Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content. The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment. Those that are not stained are called Gram-negative bacteria. Subsequently, a decolorizer, often solvent of ethanol and acetone, is used to remove the dye. Bacteria that are stained by the Gram test are called Gram-positive bacteria. The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye. The first step in gram staining is the use of crystal violet dye for the slide's initial staining. The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative ( Figure 2.34 ). The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms. Figure 2.33 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. Gram-negative bacterial cells have a thicker cell wall and require the L-ring for support. Gram-negative bacterial cells have larger flagella and require more basal body rings for support. The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. Gram-positive bacterial cells do not have an outer membrane where the L-ring is located. Often the first test performed, gram staining involves the use of crystal violet or methylene blue as the primary color. It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882, mainly to identify organisms causing pneumonia. The Gram staining is one of the most crucial staining techniques in microbiology.
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