The Gram Stain
The Gram stain is the most widely used staining procedure in bacteriology. It
is called a differential stain since it differentiates between Gram-positive and
Gram-negative bacteria. Bacteria that stain purple with the Gram-staining
procedure are termed Gram-positive; those that stain pink are said to be Gramnegative.
The terms positive and negative have nothing to do with electrical
charge, but simply designate 2 distinct morphological groups of bacteria.
Gram-positive and Gram-negative bacteria stain differently because of fundamental
differences in the structure of their cell walls. The bacterial cell wall
serves to give the organism its size and shape, as well as to prevent osmoticlysis. The material in the bacterial cell wall that confers rigidity is peptidoglycan.
In electron micrographs, the Gram-positive cell wall appears as a broad,
dense wall 20–80 nm thick and consists of numerous interconnecting layers of
Chemically, 60% to 90% of the Gram-positive cell wall is peptidoglycan
Interwoven in the cell wall of Gram-positive are teichoic acids. Teichoic
acids, that extend through and beyond the rest of the cell wall, are composed
of polymers of glycerol, phosphates, and the sugar alcohol ribitol. Some have
a lipid attached (lipoteichoic acid). The outer surface of the peptidoglycan is
studded with proteins that differ with the strain and species of the bacterium.
The Gram-negative cell wall, on the other hand, contains only 2–3 layers
of peptidoglycan and is surrounded by an outer membrane composed of
phospholipids, lipopolysaccharide, lipoprotein, and proteins. Only 10%–20% of
the Gram-negative cell wall is peptidoglycan. The phospholipids are located
mainly in the inner layer of the outer membrane, as are the lipoproteins that
connect the outer membrane to the peptidoglycan. The lipopolysaccharides,
located in the outer layer of the outer membrane, consist of a lipid portion
called lipid A embedded in the membrane, and a polysaccharide portion
extending outward from the bacterial surface. The outer membrane also contains
a number of proteins that differ with the strain and species of the bacterium.
The Gram-staining procedure involves 4 basic steps:
- The bacteria are first stained with the basic dye crystal violet. Both Grampositive
and Gram-negative bacteria become directly stained and appear
purple after this step.
- The bacteria are then treated with Gram’s iodine solution. This allows the
stain to be retained better by forming an insoluble crystal violet-iodine
complex. Both Gram-positive and Gram-negative bacteria remain purple
after this step.
- Gram’s decolorizer, a mixture of ethyl alcohol and acetone, is then added.
This is the differential step. Gram-positive bacteria retain the crystal violetiodine
complex, while Gram-negative are decolorized.
- Finally, the counterstain safranin (also a basic dye) is applied. Since the
Gram-positive bacteria are already stained purple, they are not affected by
the counterstain. Gram-negative bacteria, which are now colorless, become
directly stained by the safranin. Thus, Gram-positive bacteria appear purple
and Gram-negative bacteria appear pink.
With the current theory behind Gram-staining, it is thought that in Grampositive
bacteria, the crystal violet and iodine combine to form a larger molecule
that precipitates out within the cell. The alcohol/acetone mixture then causes
dehydration of the multilayered peptidoglycan, thus decreasing the space between
the molecules and causing the cell wall to trap the crystal violet-iodine complex
within the cell. In the case of Gram-negative bacteria, the alcohol/acetone mixture,
being a lipid solvent, dissolves the outer membrane of the cell wall and may
also damage the cytoplasmic membrane to which the peptidoglycan is attached.
The single thin layer of peptidoglycan is unable to retain the crystal violetiodine
complex and the cell is decolorized.
It is important to note that Gram-positivity (the ability to retain the purple
crystal violet-iodine complex) is not an all-or-nothing phenomenon, but a matter
of degree. There are several factors that could result in a Gram-positive organism
- The method and techniques used: Overheating during heat fixation, overdecolorization
with alcohol, and even too much washing with water
between steps may result in Gram-positive bacteria losing the crystal violetiodine
- The age of the culture: Cultures more than 24 hours old may lose their
ability to retain the crystal violet-iodine complex.
- The organism itself: Some Gram-positive bacteria are more able to retain the
crystal violet-iodine complex than others.
Therefore, one must use very precise techniques in Gram staining and
interpret the results with discretion.
Trypticase soy agar plate cultures of Escherichia coli
(a small, Gram-negative rod)
epidermidis (a Gram-positive coccus in irregular, often grapelike
The Capsule Stain
- Heat-fix a smear of a mixture of Escherichia coli and Staphylococcus epidermidis
- Using the dropper bottle of distilled water found in your staining rack,
place a small drop of water on a clean slide by touching the dropper
to the slide.
- Aseptically remove a small amount of Staphylococcus epidermidis from
the agar surface and mix it generously with the water. Flame the loop
and let it cool. Now, aseptically remove a small amount of Escherichia
coli and sparingly add it to the water. Flame the loop and let it cool.
- Using the loop, spread the mixture over the entire slide to form a thin
- Allow this thin suspension to completely air dry.
- Pass the slide (film-side up) through the flame of the bunsen burner 3
or 4 times to heat-fix.
- Stain with Hucker’s crystal violet for 1 minute. Gently wash with water.
Shake off the excess water, but do not blot dry between steps.
- Stain with Gram’s iodine solution for 1 minute and gently wash with water.
- Decolorize by adding Gram’s decolorizer drop by drop until the purple
stops flowing. Wash immediately with water.
- Stain with safranin for 1 minute and wash with water.
- Blot dry and observe using oil immersion microscopy.
Many bacteria secrete a slimy, viscous covering called a capsule or glycocalyx.
This is usually composed of polysaccharide, polypeptide, or both.
The ability to produce a capsule is an inherited property of the organism,
but the capsule is not an absolutely essential cellular component. Capsules are
often produced only under specific growth conditions. Even though not essential
for life, capsules probably help bacteria survive in nature. Capsules help many
pathogenic and normal flora bacteria to initially resist phagocytosis by the
host’s phagocytic cells. In soil and water, capsules help prevent bacteria from
being engulfed by protozoans. Capsules also help many bacteria adhere to
surfaces and, thus, resist flushing.
Skim milk broth culture of Enterobacter aerogenes
—the skim milk supplies essential
nutrients for capsule production and also provides a slightly stainable
The Capsule Stain
- Stir up the skim milk broth culture with your loop and place 2–3 loops of
Enterobacter aerogenes on a slide.
- Using your loop, spread it out over the entire slide to form a thin film.
- Let it completely air dry. Do not heat-fix. Capsules stick well to glass, and
heat may destroy the capsule.
- Stain with crystal violet for 1 minute.
- Wash off the excess stain with copper sulfate solution. Do not use water!
- Blot dry and observe using oil immersion microscopy. The organism and
the milk dried on the slide will pick up the purple dye, while the capsule
will remain colorless.
- Observe the demonstration capsule stain of Streptococcus pneumoniae (the
pneumococcus), an encapsulated bacterium that often has a diplococcus
Make a drawing of your capsule stain preparation of Enterobacter aerogenes
the demonstration capsule stain of Streptococcus pneumoniae
The Gram Stain
- Explain why the Gram stain is a differential stain.
- Describe the differences between a Gram-positive and Gram-negative cell
- Explain the theory as to why Gram-positive bacteria retain the crystal
violet-iodine complex, while Gram-negatives become decolorized.
- Describe 3 conditions that may result in a Gram-positive organism staining
- Describe the procedure for the gram stain.
- Perform a Gram stain with the necessary materials.
Determine if a bacterium is Gram-positive or Gram-negative when microscopically
viewing a Gram stain preparation, and describe the shape and arrangement of
The Capsule Stain
Describe the chemical nature and major functions of bacterial capsules.
Recognize capsules as the structures observed when microscopically viewing a
capsule stain preparation.