Gram Staining The Gram stain is a differential staining technique that differentiates nearly all bacterial species into two groups, Gram-negative or Gram-positive, based on structural differences in the cell wall. The Gram stain is a useful technique in helping to identify the presence and species of bacteria. Answer the following questions based off all the resources available. 1. What are three notable differences between Gram-negative and Gram-positive bacteria? The three notable differences between Gram-negative and Gram-positive bacteria are the size of the peptidoglycan layer, the color differences after gram staining and the absence/presence of an outer lipid membrane. - - Peptidoglycan Layer Differences : Gram-positive bacteria have a thick peptidoglycan layer whereas Gram-negative bacteria have a thin peptidoglycan layer. Color Differences: Gram-positive bacteria have a distinctive purple appearance after it has gone through the gram staining process whereas Gram-negative is known to have a pink appearance after gram staining. Outer Lipid Membrane Differences: There is a difference in the presence and absence of an outer lipid membrane. Gram-negative bacteria have a present outer lipid membrane whereas Gram-positive bacteria do not. 2. What are the four reagents of a Gram stain and what are their function during the Gram staining process? How do the chemical properties of the reagents help contribute to their function? The four reagents of a Gram stain are crystal violet, Iodine, 95% Ethyl Alcohol and Safranin. - - - Crystal Violent Function: Crystal violet is known to be the primary stain for gram staining, it is used to color both Gram-positive and Gram-negative bacteria. Iodine Function: Iodine acts as a mordant that fixes the violet stain in Gram-positive and Gram-negative bacteria. The iodine molecules bind to the crystal violet and form CV-I complex also known as an Iodine complex 95% Ethyl Alcohol Function: The ethanol is known to decolorize e Gram-negative bacteria. The alcohol helps dissolve the outer membrane that causes a disruption in the thin peptidoglycan that then allows the purple pigmented CV-I complexes to escape the Gram-negative. The purple color remains in Gram-positive bacteria due to the thick peptidoglycan layer being able to retain the CV-I complexes. Safranin Function: Safranin can be seen as a counterstain. The safranin is pink that assists with staining the Gram-negative bacteria, pink. Chemical properties of the reagents help contribute to their function because each reagent has a specific job during the gram staining of am Gram-positive and a Gram-negative bacteria Each reagent's chemical properties are needed to allow the gram staining process to be done correctly. Each chemical in the reagent contributes to their own specific function though the process of gram staining both Gram-positive bacteria and Gram-negative bacteria. Without the chemical properties in each reagent, the gram staining process can be ruined. 3. What are five troubleshooting issues that can happen when performing a Gram stain, and what false results would result? What is one thing that can be done to ensure that the Gram stain was performed properly? 1. Bacterial Smear a. The bacterial smear must be applied carefully because if a thick layer is applied, Gram-negative bacteria appear darker and could be easily mistaken for Gram-positive bacteria. 2. Fixation a. If bacteria are overheated during fixation, the cell wall will be destroyed. This results in the bacteria not being able to retain Crystal Violet. Ultimately, all bacteria will appear Gram-positive. 3. Expired culture smears a. Culture smears that are too old should not be used because the bacterial cell wall might have broken down. They will then be incapable of retaining the Crystal Violet stain resulting in all bacteria appearing Gram-Negative. 4. Under-decolorization a. If the decolorizer is washed away before it has any effect on the cell wall, the Crystal Violet molecules will not be removed from the Gram-negative cell wall. This will result in all bacteria appearing purple or Gram-positive after counterstaining with Safranin. 5. Over-decolorization a. Leaving the decolorizer on a slide for too long will damage the cell wall of both Gram-positive and Gram-negative bacteria. The Gram-positive bacteria will not be able to retain Crystal Violet. The end result will be all bacteria appearing Gram-negative or pink after counterstaining with Safranin. 6. What can be done to ensure that the Gram stain was performed properly? a. One thing that can be done to ensure that the Gram stain was performed properly is to use positive and negative controls. Positive and negative controls are bacterial smears used to test if the Gram stain was properly done. If these controls are not what was expected, then the stain was not properly performed. 4. What would most likely occur if you only applied the decolorizer for one second? The gram-negative bacteria would not decolorize properly if the decolorizer was only applied for one second. All of the bacteria would remain purple (gram-positive). The Crystal Violet molecules will not escape the Gram-negative cell wall if the alcohol (decolorizer) is rinsed away before it has any effect on the cell wall. That would result in under-decolorization. 5. When performing a Gram stain, you noticed that some bacteria on the slide appear to be purple, while other bacteria appear pink. What are three possible reasons for the results that you are seeing? ● If the bacteria cell has a cell wall that is characterized by a thick layer of peptidoglycan, the cell will reflect as purple when stained ● If the bacteria have cell walls that are characterized by a thin layer of peptidoglycan surrounded by an outer membrane,when these cells are stained they are reflected as pink ● If a bacteria cell is gram positive it will appear purple, though if it is gram negative the bacteria cell will appear pink
