Name: _______________
Section _________
Date: _______
Microbiology 197
Gram Stain Procedure
Materials required:
 Glass slide
 China marker
 Incinerator
 Bacteriologic loop
 Slide holder (clothes pin)
 Gram Stain Set
 Microscope drawing form
 Specimen: Trypticase soy agar (TSA) cultures S. aureus and E. coli
Requirements:
Prepare and fix slide from of the two specimens. Gram stain the two specimens and examine
each under oil immersion. All the necessary instructions follow below.
Slide Preparation:
1. Heat your slide. Using the china marker draw a circle about the size of a quarter in the
center of your slide
2. Place a small drop of 0.85% NaCl in the center of the drawn circle
3. Using a sterile loop lightly touch a well isolated colony.
4. Place the loop into the saline drop and spread the drop to the size of the circle.
5. Allow to air dry.
Heat fixation:
Carefully pass the air dried slide over the top of the incinerator for a count of four. Do this
three times until it is hot to the touch. Do not cook the bacteria.
Gram Stain:
1. Primary Stain: Place the slide in a slide holder. Add enough crystal violet to cover the
fixed specimen. Allow to sit for 1 minute. Rinse with water until all stain has run off.
Drain excess water from the slide.
2. Mordant: Add enough Gram’s iodine to cover the specimen. Allow to sit for 1 minute.
Rinse with water until all stain has run off. Drain excess water from the slide.
3. Decolorization: Add enough acetone/alcohol decolorizer to cover the slide. Allow to sit
for 2-3 seconds. Rinse with water. Repeat a second time. Drain excess water from the
slide. You stain will be decolorized when you see no purple color draining off the slide.
a. Critical step: Over or under decolorizing will lead to false interpretation of the
stain.
4. Counterstain: Add enough safranin to cover the specimen. Allow to sit for 1 minute.
Rinse with water until all stain has run off. Drain excess water from the slide. Wipe the
back of the slide to remove residual stain. Blot, do not wipe, excess water from the
stained side of the slide. Allow to air dry.
Examination:
Make all observations using 1000X (oil immersion). Observe the Gram reaction, cell
morphology and arrangement. Record your Gram stain analysis below. Draw what you see
and properly label the drawing. (1pt.)
Color
Morphology
Arrangement
Interpretation
S. aureus
E. coli
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Name: _______________
Section _________
Specimen: ________________
Date: _______
Observations:
Magnification: ___________
Specimen: ________________
Observations:
Magnification: ___________
1. Troubleshoot the following situations. Assume that you performed all the steps of the
procedure with the following exceptions. Each mistake should be considered separately.
Your answer should be what color a gram positive cell and what a gram negative cell
would look like if there was a: (2 pts)
Gram positive organisms
will be
Gram negative organisms
will be
a) failure to add iodine
b) failure to apply the
decolorizer
c) failure to apply the
safranin
d) reversed application of
crystal violet and safranin
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Name: _______________
Section _________
Date: _______
2. Both crystal violet and safranin are basic stains and may be used to do a simple stain as
we previously did. What step of the Gram Stain procedure makes the Gram Stain a
differential stain? (1 pt)
3. Examine the picture from your lab book (Fig. 5.7, page 39) of a gram stain from the
mouth. Look at the large epithelial cell. This is a human tissue cell. Based on your
observation, are you Gram-positive or Gram-negative? WHY? (1 pt)
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