Module 6: Gram Staining
•Differential Staining
•Reagents
•Primary stain
•Mordant
•Decolorizer
•Counterstain
•Critical issues
Objectives
To be familiar with or be able to describe:
• Difference between simple and differential staining
methods.
• Theoretical and chemical basis of Gram stain procedure.
• Relationship between Gram staining and physiology of the
microbe
• Role of chemicals used during the Gram staining
procedure
• Appearance of Gram positive cells and Gram negative
cells.
• Procedure of Gram staining technique.
• Factors that can affect the outcome of a Gram staining
procedure.
• Information that can be obtained from a Gram stained
slide about the characteristics of the microbe.
Key Words
Simple stain Differential stain
Gram reaction Cell Wall
Gram negative Gram positive
Primary stain Mordant
Decolorizer Counterstain
False positive False negative
Gram variable
Types of Staining
Simple Staining Differential Staining
Uses only one dye Uses two or more dyes
Purpose is to see Gram Stain
general Purpose is to
characteristics like distinguish between
cell shape and two categories of cells
grouping
Gram Gram
Positive Negative
Differential Staining: Gram Stain
• Gram Staining
– Type of differential staining
– Uses two contrasting stains
• Primary and Counter Stains
• Allows for differentiation between two categories of
bacteria due to the differences in their cell wall
– Gram-positive and Gram-negative.
– Gram positive bacteria appear blue/deep
purple
– Gram negative bacteria appear red pink
Differential Stains: Gram Stain
Gram positive bacteria appear blue/deep purple
Gram negative bacteria appear red pink
Figure 3.10b
Importance of Gram Staining
• Importance of Gram Staining
– Help in identification of species in microbiology lab
– Research
– Diagnosis of causative infectious agents
– Treatment of bacterial infections
– Prevention of diseases
– Disinfection of bacteria
• Cell Wall characteristics determine how bacteria are
affected by stains, antibiotics, and disinfectants
– Gram-positive bacteria tend to be more easily killed
by antibiotics like penicillin and disinfectants.
– Gram-negative bacteria are more resistant to
antibiotics and disinfectants.
Cell Wall of Bacteria
• Cell Wall Characteristics of Bacteria
– Gram positive cell wall has 40 or more layers
of peptidoglycan and is very permeable
– Gram negative cell wall has only 2-4 layers
of peptidoglycan but in addition also has an
outer membrane made up of LPS. The outer
membrane makes Gram negative wall very
impermeable
Reagents Used During Gram Staining
• Primary stain
– Crystal violet
• Mordant
– Iodine (IKI)
• Decolorizing agent
– Acetone, Alcohol, or a mixture of both
• In lab we use acetone
• Counterstain
– Safranin
Reagents Used in Gram Staining
Gram Stain
Procedure
Primary Stain
• Primary stain (Crystal Violet: Blue stain)
– Basic dye which means that it has positive
ions. This allows the dye to be attracted
towards the negatively charged cell wall of the
bacteria.
– Enters the cell and forms crystals inside the
cells
– Colors all bacteria cells purple
Mordant
• Mordant (IKI)
– Mordant is a chemical used to intensify the
color of chromophores/stains.
– Iodine binds to crystal violet crystals
inside the cell and enlarges them
– Intensifies the purple color of all cells
Decolorizing Agent
• Decolorizer: Acetone
– In Gram positive cells decolorizer dehydrates the
many layers of peptidoglycan; desiccated state shrinks
the peptidoglycan which acts as a barrier and retains
the primary stain
– In Gram negative cells decolorizer damages nonpolar
outer membrane. Dissolves the phospholipids of
membranes and makes them porous. Thin layer of
peptidoglycan cannot retain primary dye complex and
they become colorless
• Cells should have an intact cell membrane for the
decolorizing to work correctly. Older cultures usually have
a deteriorating plasma membrane
– For Gram staining only young cultures, 24-48 hours
old should be used
Counterstain
• Counterstain: Safranin, red stain
– Safranin is a basic dye and easily enters
bacteria cells
– Safranin has affinity for internal cytoplasmic
molecules
– Safranin is red pink
• Colors Gram positive cells which are blue
and makes them appear purple
• Colors the colorless Gram negative cells a
red pink color
Gram Staining Procedure
Fig. 3.14
Theory of Gram Staining
• Differentiation of bacteria as Gram Positive and
Negative possible due to differences in cell wall
characteristics.
– Gram positive bacteria cell walls have more
PTG which is permeable to solutions
– Gram negative bacteria have less PTG but
have an impermeable outer membrane made
up of lipids
• Outer membrane very impermeable but is
dissolved by organic solvents
Gram Staining Theory
• Crystal violet (CV) crystals form in cell
• Mordant (iodine) enter cells and sticks to CV crystals and
enlarges them (CV-I)
• Gram-positive cells
– Acetone dehydrates thick peptidoglycan but does not
make holes in it
– The dried PTG create a barrier and CV-I crystals remain
inside the cell giving it a purple color
• Gram-negative cells
– Acetone dissolves outer membrane because it is made
up of lipids and leaves holes
– CV-I crystals wash out and leave the cell colorless
– Secondary stain then stains them red-pink
Fig. 3.34
Gram Staining
and Cell Wall
Fig. 3.34
Gram Staining Animation
Linked directly in Canvas
Gram Stain Procedure
Color of Color of
Gram + cells Gram – cells
Primary stain: Blue/Purple Blue/Purple
Crystal violet
Mordant: Blue/Purple Blue/Purple
Iodine
Decolorizing agent: Blue/Purple Colorless
Alcohol-acetone
Counterstain: Blue/Purple Red/Pink
Safranin
Gram Positive and Negative Cells
Mixed Smear
Gram
Negative
Cells
Gram
Positive
Cells
Zoom and enlarge image to see the differences
Critical Issues
• Young cultures (24-48 hours old) should be used to
ensure that cell wall is intact
– Degeneration of cell walls happens as culture ages
– Old cells will not retain the crystal violet stain
• Smears should be even and thin, so that stain will be
evenly retained and decolorizing is even
– Gram Variable: in a smear of only one species of
bacteria some cells appear blue and some appear red
– Due to uneven decolorizing
• Fixation should not be overdone to ensure that cells are
in good condition at time of staining
• Stains should be appropriately added and timed correctly
Critical Issues
• Decolorization is the most important step
– It is the differentiation step. After this step
• Gram positive cells are deep blue
• Gram negative cells are colorless
– Over or under decolorizing should be avoided
• False positive: When Gram negative cells are under-
decolorized they will come out blue instead of red
• False negative: When Gram positive cells are over-
decolorized they will come out pink instead of blue
