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
− 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’s 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 is acidic and negatively charged:
−
−
−
Nigrosin (black dye)
Congo Red
Stains the background, not the MO
− No need for chemicals and heat fixing
− Cells appear less shriveled and
distorted – more natural
−TECHNIQUE:
1.PLACE DROP OF STAIN AT END
OF SLIDE
2.DROP OF MOs ½ INCH BEFORE
STAIN
3.WITH 2ND SLIDE HELD AT 45*,
DRAW ACROSS MOs, THEN
ACROSS STAIN
4.REVERSE DIRECTION, SMEAR
FORWARD
Bacterial cocci stained with nigrosin stain
3. DRY PREPARATIONS
• MOs are dried and killed by “FIXING”
– To flame quickly 3X
Simple
Differential
SIMPLE STAIN
− One color dye only
− EX: Crystal Violet, Methylene Blue
− Easy, fast stain method with good results
− TECHNIQUE:
1.
2.
3.
4.
5.
6.
Add the MO to slide
Air dry the MO
Fix the MO – Put through flame 3X
Flood with stain
Rinse with water
Dry for microscopic examination
DIFFERENTIAL STAIN
−
GRAM staining differentiates bacteria into 2
groups based on the differences in cell walls
−
Use two different colored dyes
−
All bacteria absorb the first stain color
−
But some lose the color when rinsed with
alcohol and are stained with a 2nd color stain
−
Results are somewhat difficult and variable
−
Named for Christian Gram – Dutch physician
DIFFERENTIAL STAIN
• GRAM (+) bacteria have peptidoglycan
in their cell walls and retain the initial
purple stain
• GRAM (–) bacteria have more lipids in
their cell wall and treatment with
alcohol dissolves the lipids and the
purple color leaks out
• The GRAM (-) bacteria are now
colorless, so a 2nd stain is needed to
color these MO’s
Gram Positive
Bacteria
Gram Negative
Bacteria
Less lipid in cell wall
More lipid in cell wall
Peptidoglycan in cell
wall
No peptidoglycan
Spore forming rods
Many intestinal rods
Many cocci
Few cocci
Tolerant to drying
Susceptible to drying
DIFFERENTIAL STAIN
−
TECHNIQUE:
1. Stain with Crystal Violet (all MO’s are purple)
2. Cover with Gram’s iodine
3. Decolorize with alcohol
4. G+ stay purple
5. G- will lose the purple dye
6. Stain with Safranin dye
(G- MO now appear red)
Gram (-)
Gram (+)
SPECIAL STAINS
− Involve special complicated methods
not for amateurs
− Used to observe special structures:
– ENDOSPORES
– FLAGELLA
– CAPSULES