Ex. 4: Gram Stain
• Chromophores
• Basic
vs. acidic dyes
Differential Stain
1. Primary stain (stains all cells on
slide)
2. Decolorizing step (removes stain
from certain types of cells)
3. Counterstain (stains the
decolorized cells)
Strongly advised:
When doing any of the microbiology labs,
make sure to carefully follow the
procedure outlined in the Materials and
Methods section . . . And any advice
given by your instructor!
From now on: read the labs before
coming to class!
Have fun!
“Little Finger Technique”
How to label a
slide
Circles on back
Other info on front
Smear Preparation
From Solid
Medium
From Liquid Medium • Transfer one loopful of H O
2
• Suspend bacterial
sediment in culture tube
by gently tapping on it.
• Transfer one loopful of
the bacterial suspension
to slide and spread out
(You may exceed the
limits of the circle)
• Air-dry and heat-fix
into circle area of slide.
• Using loop, suspend very
small quantity of colony of
interest in that drop of water
and spread the suspension
evenly. It should only appear
slightly milky. Do NOT use
too many bacteria
• Air-dry and heat-fix
Gram Stain is ….
…the most important bacterial stain!
Therefore:
Memorize steps as soon as possible
Gram Staining Reagents
Decolorizing
agent
Primary stain
Mordant
Counterstain
Gram Stain Mechanism
(slide from lecture)
• Crystal violet-iodine crystals form in cell.
• Gram-positive
– Alcohol dehydrates peptidoglycan
– CV-I crystals do not leave
• Gram-negative
– Alcohol dissolves outer membrane and leaves holes
in peptidoglycan.
– CV-I washes out
Gram Stain Animations
ASM Microbe Library
YouTube presentation
MacGraw Hill Animation
Artifacts
Precipitated crystal violet stain may resemble cocci.
Clues: dense clumping, small size of the gram-positive dots.
Paucity of organisms elsewhere in the field.
Examples of Gram Stains
Gram Stain of Urine
Gram Stain of Urine