Questions:
The main goal of a differential stain is to distinguish between gram-positive and gram-negative bacteria by utilizing distinct colors. Gram-positive bacteria will appear purple when stained, whereas gram-negative bacteria will appear pink. In contrast, a simple stain treats all microorganisms equally and enhances contrast. The key distinction between these staining techniques lies in their impact on specific microorganisms. Differential stains specifically target microbes based on differences in their cell walls, while simple stains uniformly affect all microbes.
Gram-positive and gram-negative bacteria cell walls exhibit notable disparities. Gram-positive cell walls possess a thick structure comprising numerous interconnected layers of peptidoglycan. In contrast, gram-negative cell walls have a thinner composition consisting of just one layer of peptidoglycan, which is two or three layers in thickness.
The purpose of crystal violet in the Gram’s stain procedure is to enhance the contrast of both gram-negative and gram-positive bacteria, resulting in a purple appearance.
The purpose of iodine in the Gram’s stain procedure is to form an insoluble complex with crystal violet and enhance its retention by the cell. A mordant is employed to establish or preserve a stain.
The purpose of using acetone-alcohol in the Gram’s stain procedure is to distinguish between gram-negative and gram-positive bacteria. This substance acts as a decolorizer, eliminating the stain from gram-negative bacteria but enabling gram-positive bacteria to preserve it.
The purpose of safranin in the Gram’s stain procedure is to directly stain the gram-negative bacteria, which have become decolorized. The safranin does not affect the already stained gram-positive bacteria. Overall, safranin gives the gram-negative bacteria a pinkish red color.
G.Why do gram-positive cells stain purple? Be specific
Gram-positive cells retain the crystal violet-iodine complex in their cell walls, resulting in a purple stain. This is due to the positive charge of the crystal violet dye, which is attracted to the negative charge in the cell. Additionally, the thicker peptidoglycan layer in gram-positive cells further aids in retaining the dye.
H. Specifically, gram-negative cells stain pink because they are unable to keep the crystal violet-iodine complex and thus lose their color. This happens due to their thinner peptidoglycan layer, which removes the dye when treated with acetone-alcohol. However, when safranin is applied and not rinsed off, the cell wall retains the pinkish-red dye.
I. Please indicate which of your organisms, E.Coli and yeast, stained gram-negative. Also, note if your observations align with existing knowledge about these organisms. If there are discrepancies, provide potential explanations for why the specimens may have appeared gram-negative when observed under the microscope.
J. Which organisms in your sample showed a positive gram stain? Do your observations match what is known about these organisms? If not, provide reasons why the specimen may have appeared gram positive under the microscope. The organisms that stained gram positive were Yeast, S.Epidermidis, and L.Acidopholis.
