Histology and Staining

Gram Positive Bacteria

• single cell membrane

• thick cell wall composed of peptidoglycan

Gram negative bacteria

• cells have two membranes

• in between two membranes is “ periplasmic space” which contains a layer of peptidoglycan

Heat fixing the slide makes _________________________.

makes it certain the bacteria sticks to the glass during staining

First step - Crystal Violet flooding

• Effect on Gram negative bacteria

  • Crystal violet binds to the outer membrane

• Effect on Gram positive bacteris

  • Crystal violet binds the membrane

Second Step - Lugol’s Iodine flooding

• Effects

  • forms a strong complex with bound crystal violet

  • both gram positive and negative will stain purple

Step 3 - 95% ethanol flooding - Decolorization

• Effect

  • dissolves the outer membrane of gram negative bacteria but not gram positive bacteria

  • Gram positive will stay purple

  • Gram negative will become colorless

Step 4 - Flooding with Saffranin

• Effect

  • stain both gram negative and positive bacteria

  • Gram positive will remain purple

  • Colorless Gram negative will take up the red stain