Gram Stain Procedure:
A. Slant Cultures
1. Prepare and heat-fix smears.
2. Prepare the smears of S. epidermidis and N. sicca on a second
3. Stain the slides as follows:
a. Flood the crystal violet for one
b. Pour off excess dye and wash gently
in tap water and drain the slide against a paper towel.
c. Expose the smears to Gram's iodine
for one minute by washing with iodine, then adding more iodine and leaving
it on the smear until the minute is over.
d. Wash with tap water and drain carefully.
(Do not blot.)
e. Wash with 95% alcohol for 30 seconds.
f. Wash with tap water at the
end of the 30 seconds to stop the decolorization. Drain.
g. Counterstain with 0.25% safranin
for 30 seconds.
h. Wash, drain, blot, and examine under
i. Draw the cells showing morphology,
grouping, and relative sizes. Color a few of the cells of each bacterial
species to show the Gram reaction.
j. Save these slides and the ones from
parts B & C of this exercise to use at the next lab period.
B. Broth Cultures
1. Because the smear made from the broth will be a thin smear and nearly
invisible to the naked eye even after staining, it may be advisable to
draw a ring with a felt
open on the under side of the slide to mark the
area in which the broth smear will be made. Also, when making a smear
from broth do not add a drop of water
to the slide.
2. Hear-fix the smears, Gram stain them with the above procedure,
and examine them. When focusing the broth smear use the technique
suggested for thin smears.
3. Compare the appearance of the cells in the two smears.
Go To Results of Gram Stain