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Understanding Bacterial Staining Techniques and Methods

This article provides a general discussion on bacterial staining, covering different types of stains, their chemical makeup, examples of basic and acidic dyes, staining methods, slide preparation steps, and detailed explanations of negative, simple, and differential stains. Learn how to distinguish bacterial groups using techniques such as the Gram stain.

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Understanding Bacterial Staining Techniques and Methods

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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

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