1. LECTURE: SPECIMEN PREPARATION AND STAINING
MICROBIOLOGY – ALCAMO
LECTURE: SPECIMEN PREPARATION AND STAINING

2. 1. INTRODUCTION --- Cytoplasm of bacteria lacks color
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 cytoplasm has (-) 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

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

11. 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

12. 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

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

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

17. Penicillin

18. Gram Stain