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Gram stains
Just a few to remember
Gram StainStain 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 clustersCells are very round and deeply stained1-2 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 stainMycelial 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 sizeSometimes they over-decolorize and appear Gram negative
Over-decolorized – clear areas are spores
Listeria monocytogenesGram 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 withmodified acid fast stains
ActinomycesAntler-like branching rods
Lactobacillus speciesGram 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 speciesdo not stain well with gram stain due to the lipid in the
cell wall
Kinyoun stain –AFB positive
Gram negative bacteria
Enteric Gram negative rodsPlump rectangular in shape
@ 1.4 X 2.5 - 3µm
Pseudomonasthinner than the enteric rods
@ 1 x 3 µm
Haemophilus influenzapleomorphic small faintly staining gram negative
rods
Vibrio speciescurved or comma shaped gram negative rods
Fusobacteriumdelicate fusiforme shaped faintly staining gram
negative rods
Campylobactersea gull shaped faintly staining gram
negative rods
Yersinia pestisSafety pin bipolar staining
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Neisseria speciesGram Negative DiplococciKidney bean shaped in pairs
Fastidious Gram negative rods such as the HACEK group, Brucella, Bordetella
Small cocco – bacilli rods
Legionelladoes not stain well with Gram stain
Does stain with Carbol Fuchsin used as a counterstain – pleomorphic Gram negative rod
Human cells
PMNs
Mononuclear cells
Mononuclear cells
Epithelial cells