MICROBIOLOGY – ALCAMO

LECTURE: SPECIMEN PREPARATION AND STAINING

1. INTRODUCTIONWhy?

--- 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− Quick and easy− Since no stain is used only large

dense organisms are

visible− TECHNIQUE:

1. Place drop of specimen on clean slide

2. Place cover slip over it

Simple Staining– Positively and negatively charged

molecules are attracted to each other – MO cytoplasm has (-) charge– Basic stains have (+) charge

– Crystal Violet– Methylene Blue

– Therefore: Use (+) stains to color MOs

Bacterial cocci stained with crystal violet

NEGATIVE STAIN

− EASY, FAST, GOOD FOR SIZE EVALUATION

− STAIN BACKGROUND, NOT MO CHARGE OF STAIN IS (-)

− TECHNIQUE:1. PLACE DROP OF STAIN AT END OF SLIDE2. DROP OF MOs ½ INCH BEFORE STAIN3. WITH 2ND SLIDE HELD AT 45*, DRAW

ACROSS MOs, THEN ACROSS STAIN4. REVERSE DIRECTION, SMEAR FORWARD

−TECHNIQUE:1.PLACE DROP OF STAIN AT END

OF SLIDE2.DROP OF MOs ½ INCH BEFORE

STAIN3.WITH 2ND SLIDE HELD AT 45*,

DRAW ACROSS MOs, THEN ACROSS STAIN

4.REVERSE DIRECTION, SMEAR FORWARD

3. DRY PREPARATIONS• MOs ARE DRIED (AND KILLED) BY

“FIXING– FIXING – FLAME QUICKLY 3X

Simple Differential

SIMPLE STAIN − ONE COLOR DYE ONLY− EX: CRYSTAL VIOLET, METHYLENE BLUE− EASY, FAST STAIN METHOD, GOOD RESULTS− TECHNIQUE:

1. MO TO SLIDE2. AIR DRY MO (OR WARM, NOT HOT)3. FIX – THROUGH FLAME (3X)4. STAIN5. RINSE6. DRY FOR MICROSCOPIC EXAMINATION

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

DIFFERENTIAL STAIN − TECHNIQUE:

1. CRYSTAL VIOLET (ALL MO PURPLE)2. DECOLORIZE3. G+ STAY PURPLE4. G- WILL LOSE PURPLE DYE5. SAFRANIN DYE (G- MO NOW RED)

SPECIAL STAINS − INVOLVES SPECIAL COMPLICATED

METHODS NOT FOR AMATEURS− INVOLVES SPECIAL STRUCTURES:

– ENDOSPORES– FLAGELLA– CAPSULES

Penicillin

Gram Stain