Gram stains

Just a few to remember

 Gram Stain
Stain Procedure:
– Crystal violet – primary stain for 1 min/rinse
– Gram’s iodine – mordant for 1 min/rinse
– Acetone/Alcohol – decolorizer for 5 seconds
– Safranin – counterstain for 1 min/rinse
– Gram negative (red) and Gram positive (blue)
– Assess Gram reaction and morphology
• 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
Gram Positive Bacteria
 Staphylococcus

Gram positive cocci in clusters

Cells are very round and deeply stained
1-2 µm in size
 Beta hemolytic Streptococcus
• Gram positive cocci in long chains
• Organisms are oval with variable staining
with size range of 1 – 1.5µm
 Streptococcus pneumoniae
• Gram positive cocci in pairs
• Cocci are lancet or bullet shaped /@ 1µm size
• Sometimes can visualize a capsule (clearing
around the cells)
 Viridans Streptococcus
• Gram positive cocci in short chains and
pairs – cocci around 1 – 1.5 µm in size
• Chains are not as long as Beta hemolytic
Streptococcus
 Enterococcus species
• Gram positive cocci in short chains and
pairs with size of @ 1.5 µm
• Difficult to impossible to differentiate from
viridans Streptococcus
 Yeast – Candida species
• Large Gram positive globules (@ 8 um)
with buds
• Sometimes hyphae can be seen
 Cryptococcus neoformans
Due to the capsule of C. neoformans the
stain appears somewhat like a blob
Size range from 8 – 12 µm

• India ink can be used

to visualize the capsule
 Aspergillus species Gram stain
Mycelial Fungi do not stain well with the gram stain
 Bacillus species
Gram positive rods – large , box car shaped, sometimes
spores are visible – 1.2 X 4µ in size
Sometimes they over-decolorize and appear Gram negative

Over-decolorized – clear
areas are spores
 Listeria monocytogenes
Gram positive rodshort rods, deeply stained
– size 1.2 X 2.5µm
 Diphtheroids (Corynebacteria)gram
positive pleomorphic rods in Chinese
letter forms 1.4 X 3 µm
 Nocardia species
Delicate Gram positive
branching rods - faintly staining

Nocardia stain red with

modified acid fast stains
 Actinomyces
Antler-like branching rods
 Lactobacillus species
Gram positive thin and long rods that appear
sometimes in chains
 Clostridium species
Gram positive rods with spores
@1.2 X 4 µm - 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

Kinyoun stain –
AFB positive
Gram negative bacteria
Enteric Gram negative rods
Plump rectangular in shape
@ 1.4 X 2.5 - 3µm
 Pseudomonas
thinner than the enteric rods
@ 1 x 3 µm
 Haemophilus influenza
pleomorphic small faintly staining gram negative
rods
 Vibrio species
curved or comma shaped gram negative rods
 Fusobacterium
delicate fusiforme shaped faintly staining gram
negative rods
 Campylobacter
sea gull shaped faintly staining gram
negative rods
 Yersinia pestis
Safety pin bipolar staining

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Neisseria species
Gram Negative Diplococci
Kidney bean shaped in pairs
Fastidious Gram negative rods such as the
HACEK group, Brucella, Bordetella
Small cocco – bacilli rods
 Legionella
does not stain well with Gram stain
Does stain with Carbol Fuchsin used as a counterstain –
pleomorphic Gram negative rod
 Human cells

PMNs

Epithelial cells

Mononuclear cells

Mononuclear cells