2. I. GIVE THE GRAM-STAINING REACTION AND
MORPHOLOGY (SHAPE) OF THE FOLLOWING BACTERIA.
GRAM STAIN REACTION:
MORPHOLOGY/SHAPE:
GRAM STAIN REACTION:
MORPHOLOGY/SHAPE
GRAM STAIN REACTION:
MORPHOLOGY/SHAPE
The image shows gram positive cocci
The image shows gram negative rod
The image shows gram positive bacillus
cereus
Bacillus cereus is gram-positive rod-shaped
bacilli with square ends. Occasionally may
appear gram variable or even gram-negative
with age. They are single rod-shaped or
appear in short chains. Clear cut junctions
between the members of chains are easily
Gram-positive cocci include Staphylococcus
(catalase-positive), which grows clusters,
and Streptococcus (catalase-negative),
which grows in chains
Gram-negative bacteria are found in virtually
all environments on Earth that support life.
They are characterized by their cell
envelopes, which are composed of a thin
peptidoglycan cell wall sandwiched between
an inner cytoplasmic cell
membrane and a bacterial outer membrane
3. II. INDICATE THE FUNCTION OF THE DIFFERENT REAGENTS USED IN GRAM- STAINING
Reagent Function Expected Result Expected Result
Gram- Positive Gram- Negative
Crystal Violet
Gram’s iodine
95% Alcohol
Safranin
Add Gram's iodine for 1 minute- this is a
mordant, or an agent that fixes the crystal
violet to the bacterial cell wall. Rinse
sample/slide with acetone or alcohol for 3
seconds and rinse with a gentle stream of
water. Alcohol will decolorize the sample if
it is Gram negative, removing the crystal
violet.
The basic principle of gram staining
involves the ability of the bacterial cell wall
to retain the crystal violet dye during
solvent treatment. Gram-positive
microorganisms have higher
peptidoglycan content, whereas
gram-negative organisms have higher lipid
content.
Remel Gram Decolorizer (95% Ethyl
Alcohol) is a reagent recommended for use
in qualitative procedures to differentiate
gram-negative from gram- positive
organisms. The primary stain, crystal violet,
is a basic dye which rapidly permeates the
cell wall of all bacteria, staining the
protoplast purple.
The safranin is also used as a
counter-stain in Gram's staining. In Gram's
staining, the safranin directly stains the
bacteria that has been decolorized. With
safranin staining, the gram-negative
bacteria can be easily distinguished from
gram-positive bacteria.
Gram positive bacteria stain violet due to
the presence of a thick layer of
peptidoglycan in their cell walls, which
retains the crystal violet these cells are
stained with.
Due to differences in the thickness of a
peptidoglycan layer in the cell membrane
between Gram positive and Gram negative
bacteria, Gram positive bacteria (with a
thicker peptidoglycan layer) retain crystal
violet stain during the decolorization
process, while Gram negative bacteria lose
the crystal violet stain and are instead
stained by the safranin in the final staining
process.
Conversely, the the outer membrane of Gram
negative bacteria is degraded and the thinner
peptidoglycan layer of Gram negative cells is
unable to retain the crystal violet-iodine
complex and the color is lost. A counterstain,
such as the weakly
water soluble safranin, is added to the
sample, staining it red.
A decolorizer such as ethyl alcohol or
acetone is added to the sample, which
dehydrates the peptidoglycan layer,
shrinking and tightening it. The large
crystal violet-iodine complex is not able to
penetrate this tightened peptidoglycan
layer, and is thus trapped in the cell in
Gram positive bacteria.
Gram-negative cell walls contain a high
concentration of lipids which are soluble in
alcohol. The decolorizer dissolves the
lipids, increasing cell-wall permeability and
allowing the crystal violet-iodine complex to
flow out of the cell.
The staining method uses crystal violet
dye, which is retained by the thick
peptidoglycan cell wall found in
gram-positive organisms. This reaction
gives gram-positive organisms a blue
color when viewed under a microscope.
A counterstain, such as the weakly water
soluble safranin, is added to the sample,
staining it red. Since the safranin is lighter
than crystal violet, it does not disrupt the
purple coloration in Gram positive cells.
Safranin, another positively charged basic
dye, adheres to the cell membrane. Gram
negative cells, having no dye present at
this stage of the staining process will bind
the safranin and appear pink under the
microscope. However, the decolorized
Gram negative cells are stained red.
4. III. DIFFERENTIATE GRAM-POSITIVE CELL WALL FROM
GRAM – NEGATIVE CELL WALL
Features Gram – positive Cell
Wall
Gram –negative Cell
wall
Peptidoglycan
Complexity
Teichoic Acid
Lipopolysaccharide
complexes
Endotoxin
The Gram-positive cell wall consists of many
interconnected layers of peptidoglycan and lacks an
outer membrane. Peptidoglycan prevents osmotic
lysis in the hypotonic environment in which most
bacteria live. Teichoic acids and lipoteichoic acids
are interwoven through the peptidoglycan layers.
The Gram-negative Bacteria the cell wall is composed
of a single layer of peptidoglycan surrounded by a
membranous structure called the outer membrane.
The peptidoglycan layer is non-covalently anchored to
lipoprotein molecules called Braun's lipoproteins
through their hydrophobic head.
The cell wall structure of Gram negative bacteria is
more complex than that of Gram positive bacteria.
Located between the plasma membrane and the thin
peptidoglycan layer is a gel-like matrix called
periplasmic space.
Most Gram-positive bacteria have a relatively thick
(about 20 to 80 nm), continuous cell wall (often called the
sacculus), which is composed largely of peptidoglycan
(also known as mucopeptide or murein). The
Gram-positive cell wall consists of many interconnected layers
of peptidoglycan and lacks an outer membrane.
The peptidoglycan layers of many gram-positive bacteria
are densely functionalized with anionic glycopolymers
called wall teichoic acids (WTAs). These polymers play
crucial roles in cell shape determination, regulation of cell
division, and other fundamental aspects of gram-positive
bacterial physiology.
Structure of the Gram-negative cell wall. The wall is
relatively thin and contains much less peptidoglycan
than the Gram-positive wall. Also, teichoic acids are
absent. However, the Gram negative cell wall consists
of an outer membrane that is outside of the
peptidoglycan layer.
Lipopolysaccharide (LPS) is the major component of
the outer membrane of Gram-negative bacteria.
Lipopolysaccharide is localized in the outer layer of
the membrane and is, in noncapsulated strains,
exposed on the cell surface.
Gram-negative bacteria are surrounded by a thin
peptidoglycan cell wall, which itself is surrounded by an
outer membrane containing lipopolysaccharide.
Gram-positive bacteria lack an outer membrane but are
surrounded by layers of peptidoglycan many times
thicker than is found in the Gram-negatives.
Difference between Gram-positive and
Gram-negative peptidoglycan involves the thickness of
the layers surrounding the plasma membrane.
Whereas Gram-negative peptidoglycan is only a
few nanometers thick, representing one to a few
layers, Gram-positive peptidoglycan is 30–100 nm
thick and contains many layers.
Exotoxins are usually heat labile proteins secreted by
certain species of bacteria which diffuse into the
surrounding medium. Endotoxins are heat stable
lipopolysaccharide-protein complexes which form
structural components of cell wall of Gram Negative
Bacteria and liberated only on cell lysis or death of
bacteria.
