how to gram stain

step 1

crystal violet is applied to a bacterial smear as the primary stain for 1 minute

step 2

iodine to make the dye less soluble to adhere to cell walls for 1 minute

step 3

alcohol to decolorize and wash away stain from gram negative cell walls for 2 to 30 seconds

step 4

safranin is applied as a counterstain to visualize gram-negative bacteria for 1 minute

step five

blot with bibulous paper

crystal violet effects

stains cells purple

iodine effect

cells remain purple or blue

alcohol effect

gram positive cells stay purple or blue and gram negative become colorless

safranin effect

gram positive cells stay purple and gram negative cells appear pink or red

parts of membrane in gram negative cells

LPS, outer membrane, peptidoglycan, cytoplasmic membrane

parts of membrane in gram positive cells

peptidoglycan, teichoic acid, cytoplasmic membrane

in between each step what is done

rinse away with deionized water