Gram Stains Update 2017

Gram stains
2017 UPDATE
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
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.
Cellular Material – Gram stains are
not able to differentiate many cell types. Best for
PMNs, Mononuclear cells, and epithelial cells.
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.
Gram Positive Bacteria
Staphylococcus
Gram positive cocci in clusters
Cells are very round and deeply
stained, 0,5 – 1 µm in diameter
Streptococcus
Beta hemolytic
 Gram positive cocci in chains (long chains)
 0.5 wide by 2.0 µm in length (oval)
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
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)
Enterococcus species
 Gram positive cocci in short chains and pairs
 Difficult to impossible to differentiate from viridans
Streptococcus
Yeast – Candida species
 Large Gram positive globules (@ 8 um) with buds
 Sometimes pseudo-hyphae can be seen
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
Mycelial Fungi Gram stain
Fungi stain +/- with the Gram stain
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
Listeria monocytogenes
Gram positive short rods (0.5 X 1.5 µm)
Corynebacterium spp. (diphtheroids)
gram positive rods in Chinese
letter forms and palisades (0.5 X 4 µm)
palisades
Propionibacterium acnes – small
branching Gram positive rod
Nocardia species
Delicate Gram positive
branching rods
Nocardia are red when stained
with modified acid fast stains
Actinomyces species
Antler like branching
Sulfur Granule Formation
Gardnerella vaginalis
Gram variable rods
Lactobacillus species
Gram positive long rods that appear
sometimes in chains
Clostridium species
Gram positive rods with spores
the spores can be located throughout the organism
Mycobacterium species
do not stain well with gram stain due to
the lipid in the cell wall – stain weakly
and beaded
Kinyoun AFB stain
Gram negative bacteria
Enteric Gram negative rods
(Enterobacteriaceae)
Plump rectangular in shape
(1 x 3 µm)
Pseudomonas aeruginosa
thinner than the enteric rods
0.5 X 3µm
Haemophilus influenza
pleomorphic thin faintly staining Gram
negative rods
Vibrio species
curved or comma shaped gram negative rods
Fusobacterium species –
F. nucleatum F. necrophorum
Campylobacter
sea gull shaped faintly staining gram
negative rods
Capnocytophyga spp –
pleomorphic Gram negative bacilli, variable in size and
shape
Bacteroiodes spp
Pleomorphic Gram negative bacilli, variable in length
Yersinia pestis
Safety pin bipolar staining
---
(0.5 X 1.5 µm)
Neisseria species (meningitidis and gonorrheae)
Gram Negative Diplococci
Kidney bean shaped in pairs
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Gram Stains Update 2017