Gram Stain Laboratory
PSI Biology
Name____________________________________
Purpose
Distinguish between gram-positive and gram-negative bacteria.
Background
The Gram stain technique is a useful technique for helping to distinguish and classify different types of
bacteria. This technique is used in the medical field to help doctors diagnose and treat various
diseases. The stain is a differential stain as it allows one to classify bacteria as being either Gram
positive or Gram negative in nature. Gram positive (G+) bacteria retain a violet stain used in the Gram
staining method due to having an exposed cell wall, while Gram negative (G-) bacteria do not have an
exposed cell wall and thus do not retain the violet stain. The G+ cell wall is identified by the presence
of a thick peptidoglycan layer which allows the crystal violet stain to adhere to it. The G- cell wall,
however, contains a much thinner peptidoglycan layer covered with a lipid-polysaccharide coating or
“capsule”. This capsule is often associated with more virulent disease-causing bacteria and increased
antibiotic resistance.
Gram negative bacteria can be further identified using another stain in the Gram staining method called
safranin. Safranin gives these bacteria a reddish color.
The Gram staining process consists of the following steps:
1. Application of crystal violet, the primary stain. Once exposed to the stain, all bacteria in a
sample look purple under the microscope.
2. Application of mordant, which is also known as Gram’s iodine. Once the mordant combines
with the primary crystal violet stain in the bacteria’s cell wall, a trapped crystal complex forms
within it allowing it to retain its color.
3. Application of a decolorizing agent, which washes out the primary crystal violet stain in Gram
negative bacteria.
4. Application of a secondary stain called safranin. This stain colors the Gram negative bacteria a
red or pink color.
Safety
Gloves must be worn to prevent bacterial cross-contamination.
Materials
Microscope
Microscope slides
Wax pencils
Inoculation loops
Bunsen burner
Bacterial colonies (prepared on an agar plate)
Small washbasin or bowl
Crystal violet stain
Gram’s iodine
Safranin stain
Alcohol decolorizing agent
Blotting paper
Procedure
A. Preparing a heat-fixed bacterial smear
1. Using a wax pencil, draw a small circle in the center of the microscope slide.
2. Heat the looped end of your inoculation loop in the Bunsen burner for 10 seconds. Let the
loop cool for 1 minute. Do not place the loop down while it is cooling.
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Prokaryotes & Viruses
3. Place a drop of water in the middle of your slide’s wax circle.
4. Using your cooled, sterilized inoculation loop, gently obtain a small sample of a bacterial
colony.
5. Gently mix the end of your inoculation loop into the drop of water on your slide.
6. Allow the drop of inoculated water to air dry.
7. Once dry, pass the slide, smear-side up, carefully through the Bunsen burner’s flame 3 to 4
times. The slide is now heat-fixed.
B. Staining the Bacteria
1. Over a small basin or bowl, cover the smear with crystal violet stain for 60 seconds.
2. Rinse the smear with distilled water.
3. Cover the smear with Gram’s iodine for 60 seconds.
4. Rinse the smear with distilled water.
5. Drip the alcohol decolorizing agents onto the smear carefully until the run-off becomes clear.
Do not overuse the decolorizing agent.
6. Rinse the smear with distilled water.
7. Cover the smear with the safranin stain for 45 seconds.
8. Rinse the smear with distilled water.
9. Gently dry the smear side of the slide using blotting paper. Do not wipe the slide – blot it
gently.
C. Viewing the Bacteria
1. Observe the smear at 100X.
2. Observe the smear under 1000X (oil immersion). Identify the bacteria as either Gram
positive or Gram negative and record your observations.
Analysis
1. Why is Gram staining called a differential staining technique?
2. Describe the cellular structure(s) involved in Gram staining.
3. Why was it necessary to place your inoculation loop in the Bunsen burner’s flame?
4. Why did the decolorizing solution affect the Gram negative bacteria?
5. What may have been some sources of error for the lab?
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