2. Gram Stain
Assess Gram reaction and morphology
Gram negative (red) /Gram positive (blue)
Crystal violet – primary stain for 1 min/rinse
Gram’s iodine – mordant for 1 min/rinse
Acetone/Alcohol – decolorizer for 5 secs
Safranin – counterstain for 1 min/rinse
How to judge quality of a gram stain- are the polys
blue?
If so, slide is under-decolorized.
Are the polys washed out?
If so, the slide is over-decolorized
Poor quality stains must be repeated
3. What can go wrong?
If the stain is all blue at the
end of the staining process
You did not decolorize
the slide for enough time.
If the stain is all red and washed
out appearing at the
end of the staining process
You over decolorized with the
acetone.
4. Cellular Material – Gram stains are
not able to differentiate many cell types. Best for
PMNs, Mononuclear cells, and epithelial cells.
5. Possible Artifacts in Gram
stains
Safranin
crystals
Crystal Violet crystals
When reading a Gram stain, you need to check for the uniformity of the
cell borders of the suspected organisms, there needs to be repeating/consistent
size and shape that is suggestive of a micro-organism.
9. Streptococcus
pneumoniae
Gram positive cocci in pairs – 0.5 – 1.5 µm in size
Cocci are lancet or bullet shaped with flattened sides
together
Sometimes can visualize a capsule on gram stain –
clearing around the stained organism
10. Viridans Streptococcus
Gram positive cocci in short chains and pairs
Chains are not as long as Beta hemolytic
Streptococcus (0.5 µm – 1.5 µm)
11. Enterococcus species
Gram positive cocci in short chains and pairs
Difficult to impossible to differentiate from viridans
Streptococcus
12. Yeast – Candida species
Large Gram positive globules (@ 8 um) with buds
Sometimes pseudo-hyphae can be seen
13. Cryptococcus
neoformans/gatti
Due to the capsule of
C. neoformans/gatti
the stain appears
somewhat like a blob
An India ink prep can be used
to visualize the capsule
15. Bacillus species
Gram positive rods – large , box car shaped, sometimes
spores are visible (1.0 X 4.0 µm)
Sometimes they over-decolorize and appear Gram negative
Over-decolorized
Spores