Diagnostics of infectious diseases
Lab
Gram staining
Objectives:
1. To differentiate between the two major categories of bacteria: Gram
positive and Gram negative.
2. To understand how the Gram stain reaction affects Gram positive and Gram negative
bacteria based on the biochemical and structural differences of their cell walls.
Principle:
Staining is an auxiliary technique used in microscopic techniques used to enhance the
clarity of the microscopic image.Stains and dyes are widely used in the scientific field to
highlight the structure of the biological specimens, cells, tissues etc.
The most widely used staining procedure in microbiology is the Gram stain, discovered by the
Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram
staining is a differential staining technique that differentiates bacteria into two groups: grampositives and gram-negatives. The procedure is based on the ability of microorganisms
to retain color of the stains


used during the gram stain reaction.
Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary
stain, purple.
Gram-positive bacteria are not decolorized by alcohol and will remain as purple.
After decolorization step, a counterstain is used to impart a pink color to the decolorized
gram-negative organisms.
Importance of a Gram Stain:
The Gram stain is a very important preliminary step in the initial characterization and
classification of bacteria. It is also a key procedure in the identification of bacteria based on
staining characteristics, enabling the bacteria to be examined using a light microscope. The
bacteria present in an unstained smear are invisible when viewed using a light microscope. Once
stained, the morphology and arrangement of the bacteria may be observed as well. Furthermore,
it is also an important step in the screening of infectious agents in clinical specimens such as
direct smears from a patient.
The Gram stain procedure enables bacteria to retain color of the stains, based on the differences
in the chemical and physical properties of the cell wall.
1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal
Violet in the cell wall.
Example: Staphylococcus aureus
2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye
called Safranin.
Example: Escherichia coli
Bacterial Morphology:
Bacteria are very small unicellular microorganisms ubiquitous in nature. They are micrometers
(1µm = 10-6 m) in size. They have cell walls composed of peptidoglycan and reproduce by
binary fission. Bacteria vary in their morphological features.
The most common morphologies are:
Coccus (pleural: Cocci):
Spherical bacteria; may occur in pairs (diplococci), in groups of four (tetracocci), in grape-like
clusters (Staphylococci), in chains
(Streptococci) or in cubical arrangements of eight or
more (sarcinae).
For example: Staphylococcus aureus, Streptococcus pyogenes
Bacillus (pleural: Bacilli):
Rod-shaped bacteria; generally occur singly, but may occasionally be found in pairs (diplobacilli) or chains (streptobacilli).
For example: Bacillus cereus, Clostridium tetani
Spirillum (pleural: Spirilla):
Spiral-shapedbacteria
For example: Spirillum, Vibrio, Spirochete species.
Some bacteria have other shapes such as:
Coccobacilli: Elongated spherical or ovoid form.
Filamentous: Bacilli that occur in long chains or threads.
Fusiform: Bacilli with tapered ends.
Gram Stain Mechanism:
Gram Positive Cell Wall:
Gram-positive bacteria have a thick mesh-like cell wall which is made up of
peptidoglycan (50-90% of cell wall), which stains purple. Peptidoglycan is mainly a
polysaccharide composed of
two subunits called N-acetyl glucosamine and N-
acetyl muramic acid.
As adjacent layers of peptidoglycan are formed, they are cross
linked by short chains of peptides by means of a transpeptidase enzyme, resulting in the shape
and rigidity of the cell wall. The thick peptidoglycan layer of Gram-positive organisms allows
these organisms to retain the crystal violet-iodine complex and stains the cells as purple.
Lipoteichoic acid (LTA) is another major constituent of the cell wall of Gram-positive bacteria
which is embedded in the peptidoglycan layer. It consists of teichoic acids which are long
chains of ribitol phosphate anchored to the lipid bilayer via a glyceride. It acts as regulator of
autolytic wall enzymes (muramidases: Bacterial enzymes located in the cell wall that cause
disintegration of the cell following injury or death.)
Medical Relevance of Gram Positive Cell Wall:
LTA also has antigenic properties that stimulate specific immune responses when it is released
from the cell wall after cell death. Cell death is mailnly due to lysis induced by lysozymal
activities, cationic peptides from leucocytes, or beta-lactam antibiotics.
Gram Negative Cell Wall:
Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall)
and lose the crystal violet-iodine complex during decolorization with the alcohol
counter stain Safranin, thus appearing
reddish or pink. They also have an additional outer membrane which contains
rinse, but retain the
lipids, which is separated from the cell wall by means of periplasmic space.
Medical Relevance of Gram Negative Cell Wall:
The cell wall of Gram-negative bacteria is often a virulence factor that enables pathogenic
bacteria to cause disease. The virulence of Gram-negative bacteria is often associated with
certain components of the cell wall, in particular, the lipopolysaccharide ( otherwise known as
LPS or endotoxin). In humans, LPS elicits an innate immune response characterized by
cytokine production and activation of immune system. Inflammation occurs as a result of
cytokine production, which can also produce host toxicity.
Stain Reaction:
The four basic steps of the Gram Stain are:
1) Application of the primary stain Crystal Violet (CV) to a heat-fixed smear of bacterial
culture.
solutions into CV+ and Cl – ions. These two ions
then penetrate through the cell wall and cell membrane of both Grampositive and Gram-negative cells. The CV+ ions later interacts with negatively
CV dissociates in aqueous
charged bacterial components and stains
the bacterial cells purple.
2) Addition of Gram’s Iodine.
A mordant is a
substance that increases the affinity of the cell wall for a stain
by binding to the primary stain, thus forming an insoluble complex which gets
trapped in the cell wall. In the Gram stain reaction, the crystal
violet and iodine
form an insoluble complex (CV-I) which serves to turn the smear a
Iodine (I
– or I3 –) acts as a mordant and as a trapping agent.
dark purple color. At this stage, all cells will turn purple.
3) Decolorization with 95% ethyl alcohol.

Alcohol or acetone dissolves the lipid outer membrane of
Gram negative bacteria, thus

leaving the peptidoglycan layer exposed and

increases the porosity of the cell wall.

The CV-I complex is then washed away from the thin
peptidoglycan layer, leaving Gram negative bacteria colorless.
On the other hand, alcohol has a dehydrating effect on the cell walls of Gram positive bacteria
which causes the pores of the cell wall to shrink. The CV-I complex gets tightly bound into the
multi-layered, highly cross-linked Gram positive cell wall thus staining the cells purple.
The decolorization step must be performed carefully, otherwise over-decolorization may occur.
This step is critical and must be timed correctly otherwise the crystal violet stain will be removed
from the Gram-positive cells. If the decolorizing agent is applied on the cell for too long time ,
the Gram-positive organisms to appear Gram-negative. Under-decolorization occurs when the
alcohol is not left on long enough to wash out the CV-I complex from the Gram-negative cells,
resulting in Gram-negative bacteria to appear Gram-positive.
4) Counterstain with Safranin
The decolorized Gram negative cells can
be rendered
visible with a suitable counterstain, which is usually positively charged safranin,
which stains them pink. Pink colour which adheres to the Gram positive bacteria is masked by
the purple of the crystal violet (Basic fuschin is sometimes used instead of safranin in rare
situations).
PROTOCOL
1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the
quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Gram's iodine. Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Flood slide with decolorizing agent (ETHANOL). Wait 15 seconds or add drop by drop to slide until decolorizing
agent running from the slide runs clear .
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry
with absorbent paper.
8. Observe the results of the staining procedure under oil immersion using a Brightfield microscope. At the
completion of the Gram Stain, gram-negative bacteria will stain pink/red and gram-positive bacteria will stain
blue/purple.
In a smear that has been stained using the Gram Stain protocol, the shape, arrangement and gram reaction of a
bacterial culture will be revealed. FIG. 1. shows gram-positive (blue/purple) rods and FIG. 2. shows gram-negative
(pink/red) rods.
COMMENTS AND TIPS
Comments and tips come from discussions at ASM Conference for Undergraduate Educators 2005.
The thickness of the smear used in the Gram stain will affect the result of the stain. The step that is most
crucial in effecting the outcome of the stain is the decolorizing step. Over-decolorizing will lead to an
erroneous result where gram-positive cells may stain pink to red indicating a gram-negative result, and underdecolorizing will lead to an erroneous result where gram-negative cells may appear blue to purple indicating a
gram-positive result. The degree of decolorizing required is determined by the thickness of the smear (number of
cells on the slide). The Gram stain was discussed in detail at the American Society for Microbiology Conference for
Undergraduate Educators in 2005 when this site was reviewed. The group recommends that cells be prepared with
a thin smear with no areas of clumping or inconsistency. When staining the thin smear a short decolorizing time
should be used. Some individuals will flood the slide for 15 seconds or less with decolorzing agent, while others
recommend adding decolorizing agent drop wise for 5- 15 seconds or until the color of the decolorizing agent
running from the slide no longer shows any color.
It is recommended that young, actively growing cultures be used for gram staining. An intact cell wall is required
for an accurate gram stain. Older cultures may have breaks in the cell wall and often give gram-variable results
where a mixture of pink/red cells are seen among blue/purple cells.
Using a gram stain control is recommended. On the same slide as the test culture include a sample of cells with a
known gram stain reaction to serve as a control for success in the gram stain technique.
Gram-stained bacteria should be viewed with a brightfield microscope at 1000X magnification with oil immersion. If
the smear of cells is crowded it will be difficult to note cell shape and arrangement.
When viewing slides use brightfield microscopy and adjust the brightness sufficiently to reveal the color of the
specimen.
Freshly made staining reagents are recommended. With older staining reagents, filter stains before use.
In the Gram Stain technique, two positively charged dyes are used: crystal violet and safranin. The use of the
designation “gram-positive” should not be confused with the concept of staining cells with a simple stain that has a
positive charge.
Various formulations of decolorizing agents may be used (acetone, acetone/ethanol, ethanol). Acetone is the most
rapid decolorizer followed by ethanol and then ethanol. Ethanol is recommended for student use to prevent overdecolorization of samples
When the counterstain is added this positively charged dye will replace the crystal violet dye in the gram-positive
bacteria as well as stain the gram-negative bacteria, although the presence of the mordant slows this process
considerably it is important not to overexpose cells to counterstain