Assess Gram reaction and morphology of organism
Gram negative (red) /Gram positive (blue)
Crystal violet – primary stain
Gram’s iodine – mordant
Acetone/Alcohol – decolorizer
Safranin – counterstain
How to judge quality of gram staining – is everything blue?
If so, slide is under-decolorized.
Are the polys and other cells washed out in appearance?
If so, the slide is over-decolorized
Poor quality stains must be repeated
Presence of Peptidoglycan in the cell wall (Gram positive cell wall)
traps the crystal violet making for Gram positive staining (purple)
Cellular Material – Gram stains are not able to
differentiate many cell types. Best for PMNs, mononuclear
cells, and epithelial cells. For other cell types, differential
stains must be used.
Artifacts in Gram stains
Crystal Violet crystals
When reading a Gram stain, you need to check for the uniformity of
the cell borders of the suspected organisms, there should be
repeating/consistent size and shape of the cells that is suggestive
of a micro-organism.
Streptococcus / Beta hemolytic
Gram positive cocci in long chains
Cocci are 0.5 wide by 2.0 µm in length (oval)
Gram positive cocci in pairs – 0.5 – 1.5 µm in size
Cocci are lancet or bullet shaped with flattened ends together
Sometimes can visualize a capsule on gram stain – clearing around
the stained organism
Yeast – Candida species
Large Gram positive oval globules (@ 8 um) with buds
Sometimes pseudo-hyphae can be seen
Due to the polysaccharide
capsule of C. neoformans/gatti
the yeast appears somewhat
like a round blob
An India ink prep can be used
to visualize the polysaccharide