Jack Perkins
April 13, 2015
Jayme Peacock
Engl 202C
Audience: College juniors majoring in medical-related fields
How to Perform a Gram Stain
The gram stain is the most important stain in microbiology. It is a differential stain that
divides the bacterial domain into two classes: gram positive and gram negative. The difference
between gram positive and gram negative bacteria is a result of their cell wall structure. As
Figure 1 illustrates, gram positive cells have a thick layer of peptidoglycan (polymer composed
of sugars and amino acids) with no outer phospholipid envelope while gram negative cells have a
much thinner layer of peptidoglycan covered by an outer phospholipid envelope. Identifying this
difference in cell wall composition is crucial in a clinical setting. Not only does determining the
class of a bacteria aid in the diagnosis of an infection, but it also determines the antibiotics that
can be used to eradicate the infection.
Figure 1:
Keating, Michael. “The Gram Stain.” Pennsylvania State University. Page 55. 07 April 2015.
The following instruction set will teach you how to perform a gram stain using two
species of bacteria: Escherichia coli and Micrococcus luteus. The gram stain procedure takes two
hours to complete and consists of two major processes: heat-fixing the bacteria onto a
microscope slide and staining the bacteria. Upon completion of the procedure, you will be able to
classify E. coli and M. luteus as either gram positive or gram negative.
Warning:


The bacterial species used in this experiment can cause detrimental
health effects, such as mild pneumonia and gastric irritation.
Additionally, the staining reagents used in this experiment stain skin.
o Gloves should be worn at all times when performing the gram
stain.
o If bacteria or staining reagent contaminate exposed skin,
immediately wash the infected area with soap and hot water.
The open flame of the Bunsen burner presents fire hazards.
o Before lighting the Bunsen burner, remove all combustible
materials and chemicals away from the area and tie-back any
long hair or loose clothing.
o Do not leave the open flame unattended.
o In case of a fire, sound the nearest fire alarm and evacuate the
laboratory.
The following materials are required to perform a gram stain:
• Latex Gloves
• Wire mesh platform
• Microscope slide
• Staining box
• Escherichia coli culture
• Clothespin
• Micrococcus luteus culture
• Paper towels
• Inoculating loop
• 100 ml of crystal violet
• 1 liter of distilled water
• 100 ml of iodine
• Bunsen burner
• 100 ml of alcohol
• Gas line
• 100 ml of safranin
• Safety spark lighter
• Light microscope
Heat-fixing Bacteria onto a Microscope Slide
“Fixing” bacteria onto a microscope slide prevents the bacteria from washing off the slide when
performing the staining procedure and is commonly accomplished using the open flame of a
Bunsen burner.
Lighting the Bunsen Burner
Figure 2:
1. Put on the latex gloves.
2. Connect the Bunsen burner to the
laboratory’s gas line by placing the
nozzle of the gas line into the open
end of the Bunsen burner’s
connecting tube. The set-up should
look similar to Figure 2.
3. Turn the gas line’s lever to the “on”
position. A hissing noise should be
audible, indicating that gas is
leaving the top of the Bunsen
burner.
4. Using the safety spark lighter, ignite
the gas being emitted from the
Bunsen burner so that an open flame
is created.
Transferring Water to the Microscope Slide
1. Sterilize the inoculating loop by
holding the loop over the open
flame until it glows yellow, as
shown in Figure 3.
2. Remove the inoculating loop from
the open flame and allow it to cool
for one minute.
3. Place the inoculating loop into the
distilled water and then remove it.
Water should adhere to the center of
the inoculating loop.
4. Transfer the loopful of water to the
center of the microscope slide.
5. Repeat steps 1 and 2.
Figure 3:
Transferring Bacteria to the Microscope Slide
1. Place the inoculating loop into the
E. coli culture and then remove it.
Figure 4:
E. coli should adhere to the center
of the inoculating loop.
2. Transfer the loopful of E. coli onto
the water droplet located on the
slide, as shown in Figure 4.
3. Sterilize the inoculating loop by
holding the loop over the open flame
until it glows yellow.
4. Remove the inoculating loop from
the open flame and allow it to cool
for one minute.
5. Place the inoculating loop into the
M. luteus culture and then remove it.
M. luteus should adhere to the center
of the inoculating loop.
6. Transfer the loopful of M. luteus
onto the water droplet located on the
slide.
7. Using the inoculating loop, mix the
E. coli and M. luteus cells together
in the water droplet and spread the
water across the slide.
8. Allow the slide to dry for 30 minutes.
When the water has evaporated, a thin
white film of bacteria cells should be
present on the microscope slide.
Heat-fixing the Bacteria
Figure 5:
1. With the bacteria-covered side
facing the ceiling, heat-fix the slide
(see Figure 5) by passing it over the
Bunsen burner flame five times in a
total time period of five seconds.
2. Place the slide on a paper towel,
with the bacteria-covered side facing
the ceiling.
3. Extinguish the open flame by
turning the gas line’s lever to the
“off” position.
Staining the Bacteria Cells
The differential staining technique of the gram stain consists of a primary stain, mordant,
decolorizer, and counterstain.
Primary Stain
1. Place the wire mesh platform inside of the staining box.
2. Place the heat-fixed microscope slide on top of the wire mesh platform with the
bacteria-covered side facing the ceiling.
3. Cover the bacteria with crystal violet stain and let it sit on the slide for 30
seconds.
4. Using a clothespin, pick up the slide and wash the crystal violet from the slide
into the staining box using distilled water.
5. Gently blot the distilled water from the slide using a paper towel.
Figure 6:
The crystal violet will color all of the bacteria cells
purple, as shown in Figure 6. At this point, there will
be no visible difference between the gram positive
and gram negative bacteria.
Mordant
1. Place the microscope slide on top of the wire mesh platform with the bacteriacovered side facing the ceiling.
2. Cover the bacteria with iodine and let it sit on the slide for 30 seconds.
3. Using a clothespin, pick up the slide and wash the iodine from the slide into the
staining box using distilled water.
4. Gently blot the distilled water from the slide using a paper towel.
Figure 7:
The iodine helps the crystal violet adhere more
strongly to the peptidoglycan in the cell walls, which
makes the crystal violet difficult to remove from gram
positive cell walls. As Figure 7 illustrates, there is
still no visible difference between the gram positive
and gram negative bacteria.
Decolorizer
1. Using a clothespin, hold the slide, with the bacteria-covered side facing up, at a
45° angle over the staining box.
2. Pour alcohol on the slide in a continuous stream. Stop when the alcohol running
off of the slide is colorless.
3. Wash the alcohol from the slide into the staining box using distilled water.
4. Gently blot the distilled water from the slide using a paper towel.
Figure 8:
The alcohol removes the crystal violet from the thin
peptidoglycan layer of gram negative bacteria but is
unable to remove the crystal violet from the thick
peptidoglycan layer of the gram positive bacteria. As
Figure 8 illustrates, the two classes of bacteria will
now be visually distinct: the gram positive bacteria
will remain purple while the gram negative bacteria
will be colorless.
Counterstain
1. Place the microscope slide on top of the wire mesh platform with the bacteriacovered side facing the ceiling.
2. Cover the bacteria with safranin and let it sit on the slide for 60 seconds.
3. Using a clothespin, pick up the slide and wash the safranin from the slide into the
staining box using distilled water.
4. Gently blot the distilled water from the slide using a paper towel.
5. Let the slide air dry for 15 minutes.
Figure 9:
The safranin binds to the gram negative cells and
colors them red. Safranin is unable to bind to the
gram positive cells because the binding sites are
already taken by the crystal violent molecules. As
Figure 9 illustrates, the two classes of bacteria will
now contrast even more: the gram positive bacteria
will remain purple while the gram negative bacteria
will be red. This color difference makes the two
classes of bacteria easy to identify using a light
microscope.
Now that you have completed the gram stain procedure, you will be able to classify M.
luteus and E. coli as either gram positive or gram negative by examining the slide with a light
microscope. Using a light microscope, the circular M. luteus and the rod-shaped E. coli should be
clearly visible and will look similar to Figure 10. The purple color of M. luteus indicates that it
is a gram positive bacterial species while the red color of E. coli indicates that it is a gram
negative bacterial species. This color differential not only reveals information about the
difference in cell wall structure between the two bacterial species, but also the classes of
antibiotics that can be used to kill them.
Figure 10:
https://classconnection.s3.amazonaws.com/229/flashcards/3132229/jpg/control_spot_-1_large141058E43F81380024E.jpg