Gram stains
2019 UPDATE

Gram Stain
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)

Prepare thin smear
of material onto the
glass slide
Heat fix prior to
staining

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
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 should be
repeating/consistent size and shape of the cells that is suggestive
of a micro-organism.

Gram Positive Bacteria

Staphylococcus
Gram positive cocci in random clusters
Cells are very round and deeply stained,
0.5 – 1 µm in diameter

Streptococcus / Beta hemolytic
 Gram positive cocci in long chains
 Cocci are 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 ends 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

Bacillus species
Gram positive rods – large (1.0 X 4.0 µm)
Box car shaped, sometimes spores are visible
Can over-decolorized 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 stained
with modified acid fast stains

Actinomyces species
Gram positive rods
Antler like branching
Sulfur Granule Formation

Gardnerella vaginalis
Gram variable (not quite red or blue) rods

Lactobacillus species
Gram positive long rods that appear sometimes in chains

Clostridium species
Gram positive rods with spores,
Spores will not be stained with the Gram stain
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

Gram negative bacteria

Enteric Gram negative rods
(Enterobacteriaceae)
Plump rectangular in shape(1 x 3 µm)

Pseudomonas aeruginosa
thinner and a bit longer than enteric Gram negative 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 – faintly staining
unusually shaped Gram negative rods
F. nucleatum F. necrophorum
Spindle shaped, pointed Chaining pleomorphic GNR
ends

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
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(0.5 X 1.5 µm)

Neisseria species (meningitidis and gonorrhoeae)
Gram negative diplococci
Kidney bean shaped in pairs

Yeast – Candida species
 Large Gram positive oval globules (@ 8 um) with buds
 Sometimes pseudo-hyphae can be seen

Cryptococcus neoformans/gatti
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
capsule

Mycelial Fungi on Gram stain
Fungi stain +/- with Gram stain