A fundamental laboratory technique that is introduced in general biology and microbiology courses is staining of bacterial cells on glass slides for visualization and characterization purposes. A common procedure, the Gram stain, differentiates
between bacterial species based on the chemical composition of their cell walls. The staining procedure involves applying a primary stain, crystal violet, followed by Gram’s iodine, which acts as a mordant, decolorizing with an organic solvent such as ethanol, and counterstaining with safranin. Following the procedure, gram-positive bacteria, which are more resistant to decolorization, appear purple in color while gram-negative bacteria, which are more sensitive to decolorization, appear pink.
Students encounter a number of problems
when learning how to Gram stain and view bacterial
cells. During the staining procedure, bacterial cells
tend to be washed off the slide. Students then have
difficulty locating bacterial cells on the slide,
particularly the lightly colored (pink) gram-negative
cells. Additionally, students often over-decolorize
the cells, such that gram-positive cells, which should
appear purple, are stained pink instead. This is
particularly an issue when older cultures of bacteria
are used for the staining procedure
A fundamental laboratory technique that is introduced in general biology and microbiology courses is staining of bacterial cells on glass slides for visualization and characterization purposes. A common procedure, the Gram stain, differentiates between bacterial species based on the chemical composition of their cell walls. The staining procedure involves applying a primary stain, crystal violet, followed by Gram’s iodine, which acts as a mordant, decolorizing with an organic solvent such as ethanol, and counterstaining with safranin. Following the procedure, gram-positive bacteria, which are more resistant to decolorization, appear purple in color while gram-negative bacteria, which are more sensitive to decolorization, appear pink. Students encounter a number of problems when learning how to Gram stain and view bacterial cells. During the staining procedure, bacterial cells tend to be washed off the slide. Students then have difficulty locating bacterial cells on the slide, particularly the lightly colored (pink) gram-negative cells. Additionally, students often over-decolorize the cells, such that gram-positive cells, which should appear purple, are stained pink instead. This is particularly an issue when older cultures of bacteria are used for the staining procedure
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