SlideShare uses cookies to improve functionality and performance, and to provide you with relevant advertising. If you continue browsing the site, you agree to the use of cookies on this website. See our User Agreement and Privacy Policy.
SlideShare uses cookies to improve functionality and performance, and to provide you with relevant advertising. If you continue browsing the site, you agree to the use of cookies on this website. See our Privacy Policy and User Agreement for details.
Successfully reported this slideshow.
Activate your 14 day free trial to unlock unlimited reading.
2.
Gram Stain
Assess Gram reaction and morphology of organism
Gram negative (red) /Gram positive (blue)
Crystal violet – primary stain
Gram’s iodine – mordant
Acetone/Alcohol – decolorizer
Safranin – counterstain
How to judge quality of gram staining – is everything blue?
If so, slide is under-decolorized.
Are the polys and other cells washed out in appearance?
If so, the slide is over-decolorized
Poor quality stains must be repeated
3.
Presence of Peptidoglycan in the cell wall (Gram positive cell wall)
traps the crystal violet making for Gram positive staining (purple)
4.
Prepare thin smear
of material onto the
glass slide
Heat fix prior to
staining
5.
Cellular Material – Gram stains are not able to
differentiate many cell types. Best for PMNs, mononuclear
cells, and epithelial cells. For other cell types, differential
stains must be used.
6.
Artifacts in Gram stains
Safranin
crystals
Crystal Violet crystals
When reading a Gram stain, you need to check for the uniformity of
the cell borders of the suspected organisms, there should be
repeating/consistent size and shape of the cells that is suggestive
of a micro-organism.
8.
Staphylococcus
Gram positive cocci in random clusters
Cells are very round and deeply stained,
0.5 – 1 µm in diameter
9.
Streptococcus / Beta hemolytic
Gram positive cocci in long chains
Cocci are 0.5 wide by 2.0 µm in length (oval)
10.
Streptococcus pneumoniae
Gram positive cocci in pairs – 0.5 – 1.5 µm in size
Cocci are lancet or bullet shaped with flattened ends together
Sometimes can visualize a capsule on gram stain – clearing around
the stained organism
11.
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)
12.
Enterococcus species
Gram positive cocci in short chains and pairs
Difficult to impossible to differentiate from viridans Streptococcus
13.
Bacillus species
Gram positive rods – large (1.0 X 4.0 µm)
Box car shaped, sometimes spores are visible
Can over-decolorized and appear Gram negative
Over-decolorized
Spores
14.
Listeria monocytogenes
Gram positive short rods (0.5 X 1.5 µm)
15.
Corynebacterium spp. (diphtheroids)
Gram positive rods in Chinese
letter forms and palisades (0.5 X 4 µm)
palisades
16.
Propionibacterium acnes
Small branching Gram positive rod
17.
Nocardia species
Delicate Gram positive
branching rods
Nocardia are red stained
with modified acid fast stains
18.
Actinomyces species
Gram positive rods
Antler like branching
Sulfur Granule Formation
19.
Gardnerella vaginalis
Gram variable (not quite red or blue) rods
20.
Lactobacillus species
Gram positive long rods that appear sometimes in chains
21.
Clostridium species
Gram positive rods with spores,
Spores will not be stained with the Gram stain
Spores can be located throughout the organism
22.
Mycobacterium species
Do not stain well with Gram stain due to the lipid in the
cell wall – stain weakly and beaded
34.
Neisseria species (meningitidis and gonorrhoeae)
Gram negative diplococci
Kidney bean shaped in pairs
35.
Yeast – Candida species
Large Gram positive oval globules (@ 8 um) with buds
Sometimes pseudo-hyphae can be seen
36.
Cryptococcus neoformans/gatti
Due to the polysaccharide
capsule of C. neoformans/gatti
the yeast appears somewhat
like a round blob
An India ink prep can be used
to visualize the polysaccharide
capsule
37.
Mycelial Fungi on Gram stain
Fungi stain +/- with Gram stain