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Detailed information about a lab experiment focused on gram staining, a technique used to differentiate bacteria based on their cell walls. Students will learn about the history of gram staining, the steps involved in the process, and the significance of the results. The document also covers the differences between gram-positive and gram-negative organisms, as well as the importance of using appropriate cultures for the procedure.
Typology: Lab Reports
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Section 3 Dr. Pablo Delis Student Name 9/24/
In this lab, students will become more familiar with microorganisms and the methods used for handling and observing them in the lab. Students will perform a Gram stain, become familiar with sterile techniques for working with bacteria, recognize basic bacterial forms and structures, and recognize basic protistan structures. The lab starts by introducing students to some background information on Gram staining. The Gram stain itself was developed in 1884 by Hans Christian Gram in Germany. The stain is used to differentiate organisms based on their ability to retain a certain staining compound. The technique separates bacteria into Gram-positive (Gram+) or Gram-negative (Gram-) classifications. Gram staining can also be used in medicinal applications as well. After the background information on Gram staining, the lab goes into the technique of Gram staining. The first step involves staining a fixed smear of organisms with the primary stain of crystal violet. Next, you must apply Gram's iodine stain, which is also called mordant. Mordant is a substance capable of intensifying the reaction of a specimen to a stain. The next and most important step of Gram staining is to wash the stained smear with a decolorizing agent. In most cases, 95% ethanol is used in this step. Finally, you must counterstain the smear with safranin. Gram+ organisms will
retain the purple stain of crystal violet, while Gram- will be decolorized by the alcohol and appear pinkish or red. However, if enough alcohol is exposed to the smear, it is possible that all the cells will be decolorized. Therefore, it is important to pay careful attention to the procedure to ensure accurate results. After the procedure of Gram staining is explained, the basis of the Gram stain is explained. Gram+ organisms have thicker cell walls composed of peptidoglycan and techoic acid. However, Gram- organisms have cell walls rich in lipopolysaccharides and lipoproteins. The differences in the cell walls of the organisms results in the differences of the staining. The lab goes on to explain that age of a culture is an important factor of Gram staining. As a Gram+ organism gets older, it loses its ability to retain the crystal violet. Gram stains should be performed on 18-24 hour old cultures, or actively growing cultures, to get the best results. Some organisms are gram-variable following Gram- staining. A gram-variable organism will have some cells that stain purple (Gram+) and some that stain red (Gram-). Repeated staining of the same culture at varying times will verify the existence of truly gram-variable cultures. Still, Gram variability is relatively rare. Mixed results occur from working with impure cultures or under or over decolorizing with ethanol. After the previous background information, it goes into the procedure of the lab. This includes "Colony Characterization," "Preparation of Smears and Staining Procedures," "Primitive Eukaryotic Organisms," "'Plant-like protists' = eukaryotic algae," "'Animal-like protists' = protozoans," "Fungus-like protists," and "Demonstrations."