Gram stains 2017 UPDATE

Gram StainAssess Gram reaction and morphology

Gram negative (red) /Gram positive (blue)

Crystal violet – primary stain for 1 min/rinse Gram’s iodine – mordant for 1 min/rinse Acetone/Alcohol – decolorizer for 5 secs Safranin – counterstain for 1 min/rinse

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

What can go wrong?

If the stain is all blue at theend of the staining processYou did not decolorizethe slide for enough time.

If the stain is all red and washed out appearing at the end of the staining processYou over decolorized with the acetone.

Cellular Material – Gram stains are not able to differentiate many cell types. Best for PMNs, Mononuclear cells, and epithelial cells.

Possible Artifacts in Gram stains

Safranin crystals

Crystal Violet crystals

When reading a Gram stain, you need to check for the uniformity of thecell borders of the suspected organisms, there needs to be repeating/consistent size and shape that is suggestive of a micro-organism.

Gram Positive Bacteria

Staphylococcus

Gram positive cocci in clustersCells are very round and deeply stained, 0,5 – 1 µm in diameter

StreptococcusBeta hemolytic

Gram positive cocci in chains (long chains) 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 sides

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

Yeast – Candida species

Large Gram positive globules (@ 8 um) with buds

Sometimes pseudo-hyphae can be seen

Cryptococcus neoformans/gatti

Due to the capsule of C. neoformans/gatti the stain appears somewhat like a blob

An India ink prep can be used to visualize the capsule

Mycelial Fungi Gram stainFungi stain +/- with the Gram stain

Bacillus species Gram positive rods – large , box car shaped, sometimes spores are visible (1.0 X 4.0 µm)Sometimes they over-decolorize and appear Gram negative

Over-decolorizedSpores

Listeria monocytogenesGram 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 speciesDelicate Gram positive branching rods

Nocardia are red when stained with modified acid fast stains

Actinomyces speciesAntler like branching

Sulfur Granule Formation

Gardnerella vaginalisGram variable rods

Lactobacillus speciesGram positive long rods that appear sometimes in chains

Clostridium species Gram positive rods with sporesthe spores can be located throughout the organism

Mycobacterium speciesdo not stain well with gram stain due to the lipid in the cell wall – stain weakly and beaded

Kinyoun AFB stain

Gram negative bacteria

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

Pseudomonas aeruginosathinner than the enteric rods

0.5 X 3µm

Haemophilus influenzapleomorphic thin faintly staining Gram negative rods

Vibrio speciescurved or comma shaped gram negative rods

Fusobacterium species –F. nucleatum F. necrophorum

Campylobactersea gull shaped faintly staining gram negative rods

Capnocytophyga spp – pleomorphic Gram negative bacilli, variable in size and shape

Bacteroiodes sppPleomorphic Gram negative bacilli, variable in length

Yersinia pestisSafety pin bipolar staining

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(0.5 X 1.5 µm)

Neisseria species (meningitidis and gonorrheae)Gram Negative DiplococciKidney bean shaped in pairs