gram stains update 2017
TRANSCRIPT
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