GRAM STAINING BACTERIA
Name: ____________________________
Background
With the advent and development of the microscope, humans became aware of an
otherwise unknown world. Microbiologists like Louis Pasteur soon proposed the germ
theory of disease, and Robert Koch developed his postulates for determining whether or
not a particular disease was microbial in nature and for determining the identify of the
pathogen. In the following activity, you will learn one of the fundamental procedures for
preparing bacteria for microscopic observation. Furthermore, you will become familiar
with the bacterial anatomy and physiology making this technique possible.
Materials
Glass Slides
Wooden Close Pins
Loops
Gram Positive Bacteria Samples
Gram Negative Bacteria Samples
Crystal Violet (primary stain)
Iodine Solution (the mordant)
Ethanol Decolorizer
Safranin (counter stain)
Distilled Water in Squirt Bottle
Microscope
Oil Immersion Lens Oil
Procedure
Heat Fixing the Bacterial Sample
1. Place a small drop of water on your slide.
2. Using a previously flamed but cooled loop, transfer a small amount of gram positive
bacteria from its agar surface and mix it with the water.
3. Flame the loop, let it cool again, and transfer a small amount of gram negative
bacteria from its agar surface and mix it with the water. Use the loop to spread the
mixture over the entire slide (see diagram above).
4. Allow the slide to air-dry and then pass it over the flame of a Bunsen burner 3 or 4
times (film side up) to heat fix the sample.
Staining
1. Holding your slide with a clothes pin and over a plastic staining tray flood the sample
area with crystal violet (the primary stain). Let the stains stand for 60 seconds and
then wash with water for 5 seconds to rinse off excess dye into the plastic staining
tray. Specimens should appear blue-violet with the naked eye.
2. Next, flood the sample area with iodine (the mordant). Let the stain stand for 60
seconds and then wash with water for 5 seconds to rinse off excess mordant into the
plastic staining tray. Specimens should still appear blue-violet.
3. Add the ethanol decolorizer one drop at a time until the blue-violet color is no longer
viewed from the specimen. Too much decolorization can lead to a false gram –
result, while too little can lead to a false gram + result.
4. Finally, flood the sample area with safranin (the counter stain). Let the stain stand for
60 seconds and then wash with water for 5 seconds to rinse off excess counter stain
into the plastic staining tray. Gram-positive bacteria will not incorporate the pink
safranin counter stain remaining blue-violet. Gram-negative bacteria will take on the
safranin stain and will appear pink in color.
5. Gently blot with paper towel or air dry before viewing under a microscope.
Microscopic Viewing
1. Place the specimen on the stage and focus under low power (the short lens) using
the coarse adjustment. Next, rotate to the medium power lens and use the fine tune
adjustment if needed. Then, rotate to the high power lens and again use the fine
tune adjustment if needed.
2. Rotate toward the oil immersion half way so that there is a space for you to apply the
oil immersion lens oil to the spot where the specimen is in focus. Apply a small drop
of oil on the slide and then complete the rotation of the oil immersion lens. You may
use the fine tune adjustment to bring to focus but DO NOT 1) change the focus
dramatically or 2) return to the lower power lenses.
3. When finished, clean up by wiping the oil off the oil immersion lens using lens paper.
Place the used slide on the front desk.
Lab
Sketch and label the name of each specimen viewed in the spaces below, and note
whether they are gram positive or negative.
Questions
1. What is the function of the bacteria cell wall? What is it composed of and how are
these components arranged?
2. How do gram-positive and gram-negative bacteria appear after being gram stained?
3. Diagram and describe the difference between the cell membrane and cell wall of
gram-positive and gram-negative bacteria.
4. Of what importance is the gram staining technique with regards to medicine?
5. Surf to http://www.bv229.k12.ks.us/biophilia/lysozyme.htm and describe the function
of lysozyme in organisms, and explain its interactions with gram-positive and gramnegative bateria.
6. Surf to http://www.towson.edu/~wubah/medmicro/Germ_theory.htm and list Koch’s
postulates.