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Bacterial Staining

Bacterial Staining

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Bacterial Staining

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  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 Acid Red Methylene Blue 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

  10. 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

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

  12. Gram Stain General Theory

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

  14. Proteus Staph aureus B. cereus