1. Bacterial Staining General Discussion

2. Stains All dyes are salts –Ionize Cationic Anionic Techniques –Single dyes –Multiple dyes Use

3. Chemical Makeup of Stains Benzene = organic compound Chromophore = color Auxochrome = ionization properties Benzene + Chromophore = Chromogen –Chromogen is a colored compound only Auxochrome with Chromogen allows the dye to form salt compounds that adhere to cells.

4. Examples Methylene Blue Acid Red Giemsa

5. Basic Dyes Work best in basic pH Ionizes (Cl-, SO4-) Creates (+) Cationic chromogen Attracted to (-) acidic cell components [DNA, proteins] Examples –Methylene Blue –Crystal Violet –Carbol Fuchsin –Safranin –Malachite Green CF

6. Acidic Dyes Works best in acidic pH Ionizes (Na+, K+, Ca++) Creates Anionic (-) chromogen Attracted to (+) cell components [AA] Examples –Picric Acid –Nigrosin –India Ink –Eosin Nigrosin

7. Staining Methods Negative Stain Simple Stain Differential Stains –Group Gram Stain Acid Fast Stain –Special Structures Capsule Stain Endospore Stain Flagellar Stain

8. Slide Preparation Clean slide LABEL !!! Smear in circle –Broth –Solid + H20 Air dry first Heat fix (usually) –Kill organism –Adhere to slide –Accepts dye Problems –Too thick –Wash off specimen

9. Negative Stain Acid Dye (-) chromogen Repelled by (-) cell wall Cells –Colorless –Seen against dark background

11. Simple Stain One reagent used Soak smear 30-60 seconds Rinse with H20 Background stained Bacteria stained Basic dye –(+) chromogen –(-) cell wall –Shows morphology Size Shape Arrangement Examples –MB –CF –CV

13. Differential Stains Two or more reagents Distinguish –Bacterial groups –Specific Structures Example –Gram stain –Acid Fast Stain

14. Gram Stain General Theory

15. Time Frame 1) 1 minute 2) 1 minute 3) 15 seconds 4) 1 minute Rinse with water between each step

16. ProteusStaph aureusB. cereus