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