Lab Quiz 2: Gram Stain, Capsule Stain, and Endospore Stain (Bio 286 - Microbiology) Flashcards
Hans Christian Gram
developed Gram stain in 1884
general staining technique
- primary stain… 2. mordant… 3. decolorizer… 4. secondary stain
Gram stain - primary stain
crystal violet (1 min)
Gram stain - mordant
Gram’s iodine (1 min)
Gram stain - decolorizer
acetone alcohol (5 seconds)
Gram stain - secondary stain
safranin (1 min)
appearance of gram positive cells after crystal violet in gram stain
purple
appearance of gram negative cells after crystal violet in gram stain
purple
appearance of gram positive cells after gram’s iodine in gram stain
purple
appearance of gram negative cells after gram’s iodine in gram stain
purple
appearance of gram positive cells after acetone alcohol in gram stain
purple
appearance of gram negative cells after acetone alcohol in gram stain
colorless
appearance of gram positive cells after safranin in gram stain
PURPLE
appearance of gram negative cells after safranin in gram stain
PINK
primary colorizer
stains everything
mordant
causes primary colorizer to bind tighter
gram positive cells appear purple in gram stain
because the cell wall collapses and traps the purple dye; the purple dye overpowers the pink dye from safranin to appear purple
gram negative cells appear pink in gram stain
because the acetone dissolves the outer membrane so the purple dye dissipates, allowing the safranin to turn the now colorless cells pink
variable results in gram staining
due to age of culture, variability in amounts of peptidoglycan layer
difficult species to gram stain
spirochetes (too thin to be resolved by microscopy), mycobacterium (waxy mycolic acid layer in cell wall blocking entrance of stains), intracellular bacterial parasites (difficult to stain with analine dyes)