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MICROBIOLOGY – ALCAMO

MICROBIOLOGY – ALCAMO. LECTURE: SPECIMEN PREPARATION AND STAINING . 1. INTRODUCTION. Why? --- MOs are small and transparent --- Cytoplasm of bacteria lacks color --- Stains enhance visibility. 2. WET SPECIMEN PREPARATIONS. ORGANISMS ARE NOT DRIED BEFORE HANDLING. WET MOUNT.

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MICROBIOLOGY – ALCAMO

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  1. MICROBIOLOGY – ALCAMO LECTURE: SPECIMEN PREPARATION AND STAINING

  2. 1. INTRODUCTION Why? --- MOs are small and transparent --- Cytoplasm of bacteria lacks color --- Stains enhance visibility

  3. 2. WET SPECIMEN PREPARATIONS ORGANISMS ARE NOT DRIED BEFORE HANDLING

  4. WET MOUNT • Quick and easy • Since no stain is used only large dense organisms are visible • TECHNIQUE: • Place drop of specimen on clean slide • Place cover slip over it

  5. Simple Staining • Positively and negatively charged molecules are attracted to each other • MO cytoplasmhas (-) charge • Basic stains have (+) charge • Crystal Violet • Methylene Blue • Therefore: Use (+) stains to color MOs

  6. Bacterial cocci stained with crystal violet

  7. NEGATIVE STAIN • EASY, FAST, GOOD FOR SIZE EVALUATION • STAIN BACKGROUND, NOT MO CHARGE OF STAIN IS (-) • TECHNIQUE: • PLACE DROP OF STAIN AT END OF SLIDE • DROP OF MOs ½ INCH BEFORE STAIN • WITH 2ND SLIDE HELD AT 45*, DRAW ACROSS MOs, THEN ACROSS STAIN • REVERSE DIRECTION, SMEAR FORWARD

  8. TECHNIQUE: • PLACE DROP OF STAIN AT END OF SLIDE • DROP OF MOs ½ INCH BEFORE STAIN • WITH 2ND SLIDE HELD AT 45*, DRAW ACROSS MOs, THEN ACROSS STAIN • REVERSE DIRECTION, SMEAR FORWARD

  9. 3. DRY PREPARATIONS • MOs ARE DRIED (AND KILLED) BY “FIXING • FIXING – FLAME QUICKLY 3X Differential Simple

  10. SIMPLE STAIN • ONE COLOR DYE ONLY • EX: CRYSTAL VIOLET, METHYLENE BLUE • EASY, FAST STAIN METHOD, GOOD RESULTS • TECHNIQUE: • MO TO SLIDE • AIR DRY MO (OR WARM, NOT HOT) • FIX – THROUGH FLAME (3X) • STAIN • RINSE • DRY FOR MICROSCOPIC EXAMINATION

  11. DIFFERENTIAL STAIN • TWO DIFFERENT DYES, TWO COLORS SOMEWHAT DIFFICULT, VARIABLE • EX: GRAM STAIN – IDENTIFIES DIFFERENCE IN CELL WALLS • GRAM + ‘PEPTIDOGLYCAN’ IN CELL WALL • RETAINS PURPLE STAIN • GRAM – ‘LIPID’ IN CELL WALL, • PURPLE COLORED FAT DISSOLVED, • CELL NOW COLORLESS, MUST USE 2nd STAIN TO COLOR THESE MOs

  12. DIFFERENTIAL STAIN • TECHNIQUE: • CRYSTAL VIOLET (ALL MO PURPLE) • DECOLORIZE • G+ STAY PURPLE • G- WILL LOSE PURPLE DYE • SAFRANIN DYE (G- MO NOW RED)

  13. SPECIAL STAINS • INVOLVES SPECIAL COMPLICATED METHODS NOT FOR AMATEURS • INVOLVES SPECIAL STRUCTURES: • ENDOSPORES • FLAGELLA • CAPSULES

  14. Penicillin

  15. Gram Stain

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