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GRAM STAIN: IDENTIFICATION OF
BACTERIA
STANDARDS
• 3.1.10.C, 3.1.12.C
• 3.2.10.B, 3.2.12.B
• 3.3.10.A, 3.2.12.A
• 3.3.10.B, 3.3.12.B
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INTRODUCTION
There are numerous varieties of bacteria that exist in nature. Most bacteria can be divided
into two classes, gram-negative and gram-positive, based on a differential staining
process called the Gram stain. Differences in the cell wall separate Gram-positive
bacteria, which retain a crystal violet dye used in the staining process, from Gramnegative bacteria. The differences in the cell wall also play an important role in the types
of antibiotics that will be effective against the bacteria.
Gram-positive bacteria have a membrane which is composed of two parts, the cell wall
and the cytoplasmic membrane (Fig. 1A). The cell wall is composed primarily of
peptidoglycan, a complex of linked polysaccharide chains which provide strength and
rigidity. The peptidoglycan
layer is responsible for the
Figure 1. Graphic representation of the differences
ability of the cell to retain the
between the cell walls of (A) Gram-positive and
Gram stain. Gram-positive
(B)Gram-negative bacteria.
cells also contain lipoteichoic
A.
acids (LTA) which extend
from the cytoplasmic
membrane through the
peptidoglycan layer. Both the
LTA and the peptidoglycan
are targets for antibiotics.
Gram-negative bacteria (Fig.
1B) have a cell wall which
consists of an outer
membrane, a periplasmic
space and a cytoplasmic
membrane. The
peptidoglycan layer, found in
the periplasmic space, is
much smaller, and there is no
teichoic acid present. The
outer membrane and
cytoplasmic membrane are
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B.
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Gram Stain: Identification of Bacteria
comprised of phospholipids. The outer membrane also contains lipopolysaccharides
(LPS) and porins. The porins allow small molecules, like glucose, to diffuse through the
outer membrane. The LPS are antigenic and may be targeted by certain antibiotics. A cell
wall of this type does not retain the crystal violet stain.
The Gram stain is a basic microbiology tool to visualize and classify unknown bacteria
based on cell wall type. The staining process requires a primary stain, a mordant, a
decolorizer and a counterstain. Crystal violet, a purple dye, is the primary stain. An
iodine solution is the mordant. The iodine reacts with the primary stain creating a dye
complex that is larger than the two molecules individually. A decolorizer, 95% ethanol,
dehydrates the outer membrane and peptidoglycan layer of the cell wall, creating a
matrix. The crystal violet-iodine complex is trapped by the dehydrated peptidoglycan
only in Gram-positive bacteria. Gram-negative bacteria will still have pores large enough
to release the dye complex and will appear colorless after this step. To visualize gramnegative samples, a counterstain of safranin is used. This dye will bind to the cell wall
staining it a light red to pink color.
In this experiment you are given two types of bacteria, one Gram-negative and one
Gram-positive. You will use the Gram stain technique and subsequent analysis with a
compound microscope to distinguish between these two different types of bacteria.
GUIDING QUESTIONS
•
•
•
•
How does the Gram stain identify differences between bacteria?
What are the major differences between gram-positive and gram-negative bacteria?
What are the components of the Gram stain procedure? How does each work?
Are you able to determine which bacterium is gram-positive? Gram-negative?
MATERIALS
Escherichia coli culture
Sterile inoculation loop (2)
Microscope slides (2)
Distilled water
Test tube clamp
Paper towels
Gram’s Iodine solution
Safranin O solution
Immersion oil (optional)
Bacillus cereus culture
Bunsen burner
Beaker
95% ethanol
Wax pencil
Kimwipes
Crystal Violet solution
Microscope with 100x objective
Additional bacterial cultures (optional)
SAFETY
•
•
The stains should be handled with care. Gloves and goggles are recommended
when performing the staining part of the lab.
The glass slides will be extremely hot after heat-fixing the bacteria. Be careful not
to touch the slides until they have cooled completely.
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Gram Stain: Identification of Bacteria
PROCEDURE
1. There are two glass microscope slides at each lab station. Draw a circle in the center
of each slide; this is where the bacterial sample will be placed. Using a wax pencil, label
one slide E. coli and the other B. cereus, one for each different bacterial culture.
2. Place one drop of distilled water in the center of the circle of the E. coli slide. Use the
loop end of one of the sterile inoculation loops to obtain a drop of E. coli from the culture
tube. Mix the E. coli with the drop of water on the slide; spread the liquid thinly within
the wax circle.
3. Repeat Step 2 to make the second slide with the B. cereus bacteria. Let both slides air
dry for at least 5 minutes.
4. Your instructor will give you directions on how to light the Bunsen burner. Once the
burner is lit, grasp one end of the E. coli slide with the test tube clamp, keeping the slide
culture side up. Gently move the slide back and forth through the top of the flame until
the liquid sample is dry.
5. Place the slide to cool on a paper towel on the lab bench. NOTE: Do not touch the
slide for at least 5 minutes! It will be very hot.
6. Repeat Steps 4 and 5 for the B. cereus slide.
7. You may want to wear gloves and goggles for the following steps.
8. When both slides have cooled, add 3-4 drops of the Crystal Violet solution (primary
stain) to the heat fixed bacteria on both slides (Fig. 2). Let the slides stain for 1 minute.
9. Carefully pour off the stain into the beaker provided at the lab station. Hold the slide
at a 45° angle and rinse with distilled water. Squirt the water just above the bacterial
smear and let the water flow over the sample into the beaker.
10. Drain off any excess water. Place the slides back on the paper towel and cover the
bacterial smear with Iodine solution (mordant). Let the Iodine solution sit for 1 minute.
11. Pour off any excess iodine; rinse gently with distilled water like in Step 9.
12. Still holding the slide at a 45° angle over the beaker, rinse with 95% ethanol,
allowing the ethanol to flow across the bacterial smear. Continue to rinse until no more
color is removed from the smear.
13. Rinse the slide with water to remove the ethanol. Drain the excess water.
14. Cover both smears with Safranin O solution and let the slides sit for 1 minute.
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Gram Stain: Identification of Bacteria
Figure 2. Gram stain procedure
a. Crystal violet, 1 min
b. Distilled water rinse
into beaker
c. Gram’s iodine, 1 min
d. Distilled water rinse
into beaker
e. 95% ethanol, decolorize
f. Distilled water rinse into
beaker
g. Safronin O counterstain,
1 min
h. Distilled water rinse into
beaker
i. Blot excess liquid and air
dry for 5 min
15. Rinse the slide with water and let the slides drain. Gently blot any excess liquid and
let the slide air dry for 5 minutes.
16. Use a compound microscope with a 100x objective to examine each sample.
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Figure 3. Examples of gram-positive and gram-negative bacteria.
Gram-positive bacteria
Gram-negative bacteria
REFERENCES
Background information from:
http://www.cehs.siu.edu/fix/medmicro/genmicr.htm
Graphic of Gram-positive and Gram-negative cell walls:
http://www.cehs.siu.edu/fix/medmicro/pix/walls.gif
Gram-positive and Gram-negative micrograph:
http://bioweb.uwlax.edu/bio203/s2008/jaedike_alic/staining.htm
Cavanaugh, Danny; Mark G. Keen, Ph.D.; Department of Microbiology, North Carolina State University;
The Gram Stain: An Animated Approach:
http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/images/gram_stain_11.swf
CREDITS
Special thanks to Chris Cassano of Wilmington High School, New Wilmington, PA , and
Casey Schmidt, Science in Motion lab assistant, for testing, editing and reviewing this
protocol. The lab was revised and adapted from the above references by Dr. Stephanie
Corrette-Bennett.
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Gram Stain: Identification of Bacteria
DATA SHEET
Name: _______________________
Group: _______________________
Date: _______________________
DATA ANALYSIS
Draw a picture of what you observe for each type of bacteria. Identify which you think is
Gram-positive and Gram-negative, based on your results.
Bacillus cereus
Escherichia coli
Gram ______
Gram ______
QUESTIONS
1. Describe how the two bacteria look different after the Gram stain (size, shape, stain
color).
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Gram Stain: Identification of Bacteria
2. List the three differences between the cell walls of Gram-positive and Gram-negative
bacterial cells.
a.
b.
c.
3. What are the 4 reagents in the Gram stain procedure?
a.
b.
c.
d.
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