Performing a Gram Stain
||Step in the Staining
|0. Start with a heat-fixed slide
of your material. Recall, Gram positive bacteria have a single cell
membrane and hardy outer cell wall made of peptidoglycan. In contrast.
Gram negative cells have two membranes. In between the two membranes is
the *periplasmic space* which contains a layer of peptidoglycan.
<-- Follow the images to the right to see how Gram positive and Gram negative cells would appear during each stage of the stain.
|1. Crystal Violet: Flood the
slide with Crystal violet. Let sit for 30 seconds, then rinse with tap
water. If available, use forceps to hold the slide while rinsing. The
crystal violet binds to the membrane of Gram positives, and the
outer-membrane of Gram negative bacteria.
|2. Lugol's Iodine: Now flood the
slide with the Lugol's iodine, which forms a strong complex with bound
crystal violet. Let sit for 30s, then rinse the slide with tap water. A
this stage both Gram positive and Gram negative bacteria will stain
|3. Decolorization -- this is the
most important step. Ethanol tends to be more forgiving than acetone,
and may be worthwhile to try for beginners. Acetone or 95% ethanol
dissolves the outer membrane of Gram negative bacteria, but not Gram
positive bacteria. If done properly, Gram positive bacteria remain
purple at this stage, while Gram negative bacteria become colorless.
However, too much decolorization and all bacteria will appear Gram
negative (all crystal violet+iodine will be washed away); too little,
and all bacteria will appear Gram positive (not enough to remove the
outer membrane of Gram negatives). In general, hold the slide at a
slant and count for 3 seconds while squirting top edge of the slide
with the decolorizing agent, so it runs down the length of the slide.
Then immediately rinse in tap water to remove remaining decolorzing
|4. Counterstain with Saffranin:
Flood the slide with the dye and let sit for 30s, then wash. Saffranin
is a red dye and actually stains both Gram positive and negative
bacteria. However, for Gram positive bacteria that are already purple,
they remain an intense purple color while previously colorless Gram
negative bacteria take up the red stain and will appear red by
||5. Interpretation (more below):
Slides need to be read at 100X magnification on a microscope. Lenses at
this magnification require immersion oil to obtain clear imaging of
material on the slide. Gram positive organisms will be dark purple,
while Gram negative will appear bright red.