1. Gram staining

2. Types of staining Simple –Bacteria stained to contrast environment Differential –Bacteria differing chemically react differently to staining

3. Gram stain Differential –Gram positive = purple–Gram negative = pink

4. Fixation Add 1 drop of deionized water Burn bacteria to slide Step 1

5. Primary stain –Basic dye crystal violet 30 seconds Rinse (DI water) 5 seconds

6. Step 2 Mordant –Iodine Helps dye interact with bacteria –Dye tightly bond to cell 60 seconds Rinse 5 seconds

7. Step 3 Decolorizer –Ethanol Gram (+) bacteria retain crystal violent-iodine dye when decolorized Gram (-) bacteria lose crystal violent-iodine dye 20 seconds Rinse 5 seconds

8. Cell Wall Peptidoglycan –Polymer of cell wall –Gram (+) 20 to 80 nm thick –Gram (-) 2 to 7 nm thick Lipopolysaccharide (fats sugar) & Proteins

9. Step 4 Counterstain Safranin –Purple color of Gram (+) bacteria not altered –Colorless Gram (-) bacteria turn pink 70 seconds Rinse 5 seconds

10. Gram (+) = Deep purple Gram (-) = pinkish 1)Heat Affix Bacteria to slide – 1 drop of H2O 2)Crystal violet Stain = 30 seconds 3)Iodine Stain 60 seconds 4)Ethanol Stain 20 seconds 4)Safranin Stain 70 seconds Rinse 5 seconds E. Coli M. Luteus B. megasterium R. Rurum S. aurantaca S. Marcescens