Gram stains 2021
Margie A. Morgan, PhD. D(ABMM)

Gram Stain
Assess for 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

Prepare a thin smear of material onto the glass slide.
Air dry and then heat fix specimen on slide prior to staining.
Stain approximately one minute per step and rinse with sterile
water between each step. Air dry before scope reading.

Presence of large amount of peptidoglycan in the Gram positive cell wall,
traps the crystal violet making for Gram positive staining with the primary
stain crystal violet and the organism appearing blue in color.

Cellular Material – Gram stains are not able to differentiate
many cell types. Best for identifying polymorphonuclear leukocytes
(PMNs), mononuclear cells, and epithelial cells. For other cell types,
differential stains must be used, such as Wright-Giemsa)

Artifacts in Gram stains
Safranin
crystals
Crystal Violet crystals
When reading a Gram stain, check for the uniformity of the cell borders
of suspected organisms, there should be repeating/consistent size and
shape of the cells that is supportive of a micro-organism. Artifacts such
as stain crystals are usually not consistent in shape or size.

The most common
Gram Positive Bacteria

Staphylococcus species
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 chains of variable length
 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 – which appears as
clearing around the stained organism

Viridans Streptococcus
 Gram positive cocci in short chains and pairs
 Chains are usually not as long as the Beta hemolytic
Streptococcus, size is 0.5 µm – 1.5 µm.

Enterococcus species
 Gram positive cocci in short chains and pairs
 Can be difficult to differentiate from viridans Streptococcus, size is
also size is 0.5 µm – 1.5 µm.

Bacillus species
Gram positive rods – large (1.0 X 4.0 µm)
Box car shaped, sometimes spores are visible. Spores will not
stain with gentian violet.
Can be over-decolorized and appear Gram negative on Gram stain
Over-decolorized Spores

Listeria monocytogenes
Gram positive short rods (0.5 X 1.5 µm)
Do not appear in chains and they do not branch.

Corynebacterium species
Gram positive rods in that group in Chinese
letter forms and palisade cell arrangement (0.5 X 4 µm)
Palisade cell
arrangement

Cutibacterium (Propionibacterium)
acnes
Small pleomorphic Gram positive rods that branch.

Nocardia species
Delicate Gram positive
branching rods, sometimes
staining quite poorly and
appearing speckled.
Nocardia stain red
with modified acid fast stains

Actinomyces species
Gram positive rods with antler like branching, can be seen in sulfur
granules in tissue
Sulfur Granule
Formation

Gardnerella vaginalis
Gram variable (not quite red or blue) rods
Associated with “clue cells” in bacterial vaginosis

Lactobacillus species
Gram positive rods, thin, appear sometimes in short chains

Clostridium species
Gram positive rods, large, with spores sometimes visible.
Spores will not stain with the Gram stain and appear as colorless areas
in the cell.
Spores are located throughout the length of the rod-shaped organism

Mycobacterium species
Do not stain well with Gram stain due to lipid in the cell
wall, they stain beaded and barely visible.
Acid Fast stains are superior for the staining of
Mycobacteria

Gram negative bacteria

Enteric Gram negative rods
(Enterobacterales)
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 gram negative rods, thin and faintly staining

Vibrio species
curved or comma shaped Gram negative rods

Fusobacterium species – faintly staining
unusually shaped Gram negative rods
F. nucleatum F. necrophorum
Spindle shaped, with pointed Chaining pleomorphic rods
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 like with 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) that produce
a smaller attached cell known as a bud
 Sometimes pseudo-hyphae can be seen which are
extensions of the yeast cells into long hyphae like forms

Cryptococcus neoformans/gatti
Due to the polysaccharide
capsule of C. neoformans/gatti
the yeast appears somewhat
like a round blob on Gram
stain
 An India ink prep can be used
to visualize the polysaccharide
capsule

Mycelial Fungi
Variably staining of hyphae on Gram stain